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Contains fulltext : 49700.pdf (Publisher’s version ) (Open Access) Plasmacytoid dendritic cells (pDCs) contribute to innate antiviral immune responses by producing type I interferons. Although human pDCs can induce T cell responses upon viral infection, it remains unclear if pDCs can present exogenous antigens. Here, we show that human pDCs exploit FcgammaRII (CD32) to internalize antigen-antibody complexes, resulting in the presentation of exogenous antigen to T cells. pDCs isolated from melanoma patients vaccinated with autologous monocyte-derived peptide- and keyhold limpet hemocyanin (KLH)-loaded dendritic cells, but not from nonvaccinated patients or patients that lack a humoral response against KLH, were able to stimulate KLH-specific T cell proliferation. Interestingly, we observed that internalization of KLH by pDCs depended on the presence of serum from vaccinated patients that developed an anti-KLH antibody response. Anti-CD32 antibodies inhibited antigen uptake and presentation, demonstrating that circulating anti-KLH antibodies binding to CD32 mediate KLH internalization. We conclude that CD32 is an antigen uptake receptor on pDCs and that antigen presentation by pDCs is of particular relevance when circulating antibodies are present. Antigen presentation by pDCs may thus modulate the strength and quality of the secondary phase of an immune response.

Contains fulltext : 48814.pdf (Publisher’s version ) (Open Access) Ex vivo generated cancer vaccines based on dendritic cells (DCs) are currently applied in the clinic. The migration of DCs from the tissues to the lymph nodes is tightly controlled and involves many different mediators and their receptors. A recent study demonstrated that the rate of migration of antigen-bearing DCs in situ from the skin to the lymph node is 100-fold higher than previously estimated. The migration of ex vivo generated DCs is rather inefficient but can be improved by pre-conditioning of the vaccine injection site with inflammatory cytokines. An alternative approach that is currently being explored is to target tumor antigens directly to DCs in situ, thereby exploiting the intricate migratory capacity of DCs in vivo. Recent advances have been made in understanding DC migration in the context of DC-based vaccines.

Contains fulltext : 48743.pdf (Publisher’s version ) (Open Access) The success of cellular therapies will depend in part on accurate delivery of cells to target organs. In dendritic cell therapy, in particular, delivery and subsequent migration of cells to regional lymph nodes is essential for effective stimulation of the immune system. We show here that in vivo magnetic resonance tracking of magnetically labeled cells is feasible in humans for detecting very low numbers of dendritic cells in conjunction with detailed anatomical information. Autologous dendritic cells were labeled with a clinical superparamagnetic iron oxide formulation or (111)In-oxine and were co-injected intranodally in melanoma patients under ultrasound guidance. In contrast to scintigraphic imaging, magnetic resonance imaging (MRI) allowed assessment of the accuracy of dendritic cell delivery and of inter- and intra-nodal cell migration patterns. MRI cell tracking using iron oxides appears clinically safe and well suited to monitor cellular therapy in humans.

Dendritic cells (DC) are the most potent antigen-presenting cells and form a promising new treatment modality. Fully activated DC loaded with antigen are very useful in stimulating immune responses, in particular those to combat cancer. Immature DC can either cause immunological tolerance or induce regulatory T-cells, opening up future application in transplantation, autoimmunity, and perhaps chronic inflammation. For these clinical applications, generation of DC under Good Manufacturing Practice conditions and without the use of animal products is a main prerequisite. To date, there are many different DC culture protocols, which subsequently lead to different types of DC. Protocols differ not only in DC preparation techniques, but also many other variables, like maturation status, dose and timing interval, route of administration, and antigen loading. DC vaccination, although very promising, is far from standardized. In this chapter we discuss the usefulness of standardized clinical and immunological criteria and the need for careful study design to further optimize the use of dendritic cells and to unequivocally prove their efficacy.

Dendritic cells (DC) are the most potent antigen-presenting cells and form a promising new treatment modality. Fully activated DC loaded with antigen are very useful in stimulating immune responses, in particular those to combat cancer. Immature DC can either cause immunological tolerance or induce regulatory T-cells, opening up future application in transplantation, autoimmunity, and perhaps chronic inflammation. For these clinical applications, generation of DC under Good Manufacturing Practice conditions and without the use of animal products is a main prerequisite. To date, there are many different DC culture protocols, which subsequently lead to different types of DC. Protocols differ not only in DC preparation techniques, but also many other variables, like maturation status, dose and timing interval, route of administration, and antigen loading. DC vaccination, although very promising, is far from standardized. In this chapter we discuss the usefulness of standardized clinical and immunological criteria and the need for careful study design to further optimize the use of dendritic cells and to unequivocally prove their efficacy.

We have investigated the capacity of immature and mature monocyte-derived DCs pulsed with melanoma-associated peptides (gp100 and tyrosinase) to induce a primary cytotoxic T-lymphocyte response in vivo.Advanced HLA-A2.1(+) melanoma patients were vaccinated with peptide- and keyhole limpet hemocyanin (KLH)-pulsed DCs, either immature (9 patients) or matured by monocyte-conditioned medium/tumor necrosis factor alpha/prostaglandin E(2) (10 patients).All patients vaccinated with mature DCs showed a pronounced proliferative T-cell and humoral response against KLH. By contrast, KLH responses were absent in most of the patients vaccinated with immature DCs. Delayed-type hypersensitivity (DTH) reactions against antigen-pulsed DCs were only observed in patients vaccinated with mature DCs and not in patients vaccinated with immature DCs. MHC-peptide tetramer staining of DTH-derived T cells revealed the presence of specific T cells recognizing the melanoma-associated peptides in 1 patient. In a second patient, DTH-derived T cells showed specific lysis of tumor cells expressing the antigens used for DC pulsing. Only patients vaccinated with mature DCs showed objective clinical responses. Interestingly, both patients with long-term progression-free survival (22 and40 months) were both vaccinated with mature DCs and demonstrated antigen-specific T-cell reactivity of DTH-derived T cells.We conclude that mature DC are superior to immature DC in the induction of immunological responses in melanoma patients, which may translate into improved clinical results.

Dendritic cells are the professional antigen-presenting cells of the immune system. To induce an effective immune response, these cells should not only express high levels of MHC and costimulatory molecules but also migrate into the lymph nodes to interact with naïve T cells. Here, we demonstrate that in vitro-generated mature, but not immature dendritic cells, efficiently migrate into the T-cell areas of lymph nodes of melanoma patients. This difference is confirmed by in vitro studies, in which immature dendritic cells are strongly adherent, whereas mature dendritic cells remain highly motile. Our present findings demonstrate that the ability of dendritic cells to mount a proper immune response correlates with their ability to migrate both in vitro and in vivo.

Contains fulltext : 142347.pdf (Publisher’s version ) (Open Access) Dendritic cells (DC) that express the type II C-type lectin DC-SIGN (CD209) are located in the submucosa of tissues, where they mediate HIV-1 entry. Interestingly, the pathogen Candida albicans, the major cause of hospital-acquired fungal infections, penetrates at similar submucosal sites. Here we demonstrate that DC-SIGN is able to bind C. albicans both in DC-SIGN-transfected cell lines and in human monocyte-derived DC. The binding was shown to be time- as well as concentration-dependent, and live as well as heat-inactivated C. albicans were bound to the same extent. Moreover, in immature DC, DC-SIGN was able to internalize C. albicans in specific DC-SIGN-enriched vesicles, distinct from those containing the mannose receptor, the other known C. albicans receptor expressed by DC. Together, these results demonstrate that DC-SIGN is an exquisite pathogen-uptake receptor that captures not only viruses but also fungi.

Contains fulltext : 108371.pdf (Publisher’s version ) (Open Access) In order for cellular therapeutics to succeed, comprehensive monitoring of the transplanted cells in vivo is required i.e., their localization, functionality and numbers in a longitudinal manner. Recently, dendritic cell based vaccines have been monitored by their effect on lymphocyte activation using [(18)F]FLT PET in cancer patients.

Contains fulltext : 153217.pdf (Publisher’s version ) (Open Access) Autologous dendritic cell (DC) therapy is an experimental cellular immunotherapy that is safe and immunogenic in patients with advanced melanoma. In an attempt to further improve the therapeutic responses, we treated 15 patients with melanoma, with autologous monocyte-derived immature DC electroporated with mRNA encoding CD40 ligand (CD40L), CD70 and a constitutively active TLR4 (caTLR4) together with mRNA encoding a tumor-associated antigen (TAA; respectively gp100 or tyrosinase). In addition, DC were pulsed with keyhole limpet hemocyanin (KLH) that served as a control antigen. Production of this DC vaccine with high cellular viability, high expression of co-stimulatory molecules and MHC class I and II and production of IL-12p70, was feasible in all patients. A vaccination cycle consisting of three vaccinations with up to 15x106 DC per vaccination at a biweekly interval, was repeated after 6 and 12 months in the absence of disease progression. mRNA-optimized DC were injected intranodally, because of low CCR7 expression on the DC, and induced de novo immune responses against control antigen. T cell responses against tyrosinase were detected in the skin-test infiltrating lymphocytes (SKIL) of two patients. One mixed tumor response and two durable tumor stabilizations were observed among 8 patients with evaluable disease at baseline. In conclusion, autologous mRNA-optimized DC can be safely administered intranodally to patients with metastatic melanoma but showed limited immunological responses against tyrosinase and gp100.

Dendritic cells (DC) have been recognized as the most potent antigen presenting cells (APC) of the immune system. We performed a phase 1 study in twelve patients with metastatic renal cell carcinoma (RCC) using autologous immature DC loaded with autologous tumorlysate (TuLy) as a vaccine based on our earlier in vitro observations that such DC can activate tumor-specific cytotoxic T-lymphocytes. The treatment was combined with low-dose interleukin (IL)-2, as this has shown benefit in DC-based therapies. Patients received three intradermal vaccinations at two weekly intervals, and, after each vaccination, IL-2 was administered for 5 consecutive days. In six patients, keyhole-limpet hemocyanin (KLH) was added to the DC culture for immunologic monitoring purposes. In general, DC phenotype was CD14(low), CD86(high), CD40(high), CD80(low), and CD83(low). We noticed that the number of CD14+ cultured DC increased during treatment. Nevertheless, ovalbumin uptake remained high, underlining that these cells were still functional immature DC. The vaccine was able to elicit cellular anti-KLH responses, emphasizing the ability of the injected DC to mount an immunologic response. However, proliferative responses against TuLy were not detected, and humoral responses against TuLy or KLH were absent. Objective clinical responses were not observed, but extended stable disease was noted. The absence of cellular, humoral, or clinical antitumor responses suggests that the vaccination strategy with immature DC has little benefit for patients with advanced RCC. Nevertheless, this study shows the feasibility of a completely autologous DC and tissue culture methodology for the generation of TuLy pulsed DC.

Contains fulltext : 223818.pdf (Publisher’s version ) (Open Access) We previously identified an HLA-A2.1-restricted epitope within the RCC-associated antigen G250 that is recognized by CTLs. Using DCs of healthy individuals, which were loaded with overlapping 20 mer G250-derived peptides, we here report the induction of CD4(+) T cells that recognize the G250 peptide of amino acids 249-268. Moreover, naturally processed G250 protein is readily recognized by these G250-specific CD4(+) T cells in the context of HLA-DR molecules. Interestingly, peptide G250:249-268 overlaps the previously identified HLA-A2.1-restricted G250 epitope recognized by CTLs. These data and the high prevalence of G250 in RCC patients make peptide G250:249-268 a potential target in peptide-based vaccines to induce both CD4(+) and CD8(+) T-cell responses in patients.

Platinum-based drugs form the corner stone of many treatment schedules in cancer. Their main mechanism of actions is the covalent binding to DNA, leading to the formation of platinum-DNA adducts and thereby inducing apoptosis. Recently, other so called off-target effects of these drugs have been described. These include the induction of immunogenic cell death and the downregulation of inhibitory molecule Programmed Death-Ligand 2 by inactivation of signaling molecule/transcription factor STAT6, a member of the Signal Transducer and Activator of Transcription (STAT) protein family. STAT proteins play crucial roles in the pathogenesis and progression of cancer and much research is being conducted to identify inhibitors of STAT signaling for use in cancer treatment. Here, we investigated the potential of platinum-based chemotherapeutics to inhibit STAT signaling. We found that treatment of tumor cells of various origins with platinum drugs inhibit the activation of STAT proteins and leads to loss of cytokine-induced and constitutive STAT protein phosphorylation. Concurrently, a decrease of STAT protein nuclear accumulation was evident by confocal microscopy analysis. This resulted in a downregulation of STAT-regulated tumorigenic genes such as Bcl-xL, MCL-1, VEGF and survivin. Non-STAT target genes were not affected. Analysis of the known STAT inactivation pathways showed that these were not activated nor enhanced by treatment with platinum drugs. Finally, using a biochemical alphascreen-based assay, we demonstrated that platinum compounds directly interact with STAT proteins and inhibit binding to the SH2 domain. These findings provide novel insight into the mechanism of action of platinum drugs and show for the first time that these clinically used compounds directly affect important cellular signaling pathways. These results indicate that part of the efficacy of these drugs may be derived from their inhibition of STAT signaling and they provide important leads for the development of novel innovative treatment schedules in cancer that exploit the STAT-inhibiting effect of platinum drugs, for example by potentiating targeted therapies or immunotherapy. Citation Format: W. Joost Lesterhuis, Stanleyson V. Hato, Carl G. Figdor, Susumu Takahashi, Cornelis J.A. Punt, Akira Asai, I. Jolanda M. De Vries. Platinum-based cancer chemotherapeutics inhibit STAT signaling by blocking the SH2 domain. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2120. doi:10.1158/1538-7445.AM2013-2120

Antigen-specific immunotherapy has recently progressed through the development of effective therapeutic vaccinations in advanced melanoma (1,2) and prostate cancer (3). Antigen-specific immune responses in cancer patients can also be induced by exploiting autologous dendritic cells (DCs) that are “educated” ex vivo; i.e., DCs that are appropriately activated and loaded with tumor antigens. DCs are the most potent antigen-presenting cells of the immune system and play a central role in the induction and maintenance of antigen-specific immunity (4). These cells capture and process antigen and migrate to the lymph nodes (LNs), where they present the antigen to the adaptive arm of the immune system, inducing antigen-specific T- and B-cell responses. The detection of vaccine-induced immune responses in vivo using a clinically applicable means is critical for the optimization of novel immunotherapies. Positron emission tomography (PET) is a widely available, highly sensitive imaging modality for the in vivo visualization and quantification of molecular processes at a cellular level. Furthermore, whole body imaging allows localization in a longitudinal fashion. These features are necessary to measure immune responses by quantification of the low numbers of proliferating T and B cells early after vaccination in relevant LNs. Thus far, investigators have mainly exploited [18F]-labeled fluoro-2-deoxy-2-d-glucose ([18F]FDG) for PET imaging of proliferating cells, based on the increased glucose metabolism of these cells. Recently, novel tracers have been developed that facilitate imaging of other cellular processes: [18F]-labeled 3α-fluoro-3α-deoxy-thymidine ([18F]FLT) was designed as a tracer for cell proliferation (5) and is increasingly being applied in oncology. However, it has been recognized that enhanced nucleoside demand is not restricted to tumor cells (6). A successful vaccination results in the proliferation of activated lymphocytes in a highly controlled manner within LNs. This proliferation is accompanied by a large metabolic switch in lymphocytes and could serve as a marker of immune responsiveness. We demonstrate that [18F]FLT PET can be used to directly monitor antigen-specific immune responses in vivo shortly after therapeutic vaccination, because it offers a sensitive tool to study the kinetics and localization of induction of antigen-specific lymphocyte activation upon vaccination with antigen-loaded autologous DCs (7). Melanoma patients with lymph node (LN) metastases received DC vaccine therapy, injected intranodally, followed by [18F]FLT PET at varying time points after vaccination. Control LNs received saline or DCs without antigen. De novo immune responses were readily visualized in treated LNs early after the prime vaccination, and these signals persisted for up to 3 weeks. This selective [18F]FLT uptake was markedly absent in control LNs, although tracer uptake in treated LNs increased profoundly with as little as 4.5 × 105 DCs. Immunohistochemical staining confirmed injected DC dispersion to T-cell areas and resultant activation of CD4+ and CD8+ T cells. The level of LN tracer uptake significantly correlates to the level of circulating antigen-specific IgG antibodies and antigen-specific proliferation of T cells in peripheral blood. Furthermore, this correlation was not observed with [18F]FDG. Therefore, [18F]FLT PET offers a sensitive tool to study the kinetics, localization, and involvement of lymphocyte subsets in response to vaccination. Recently published large phase III trials, demonstrate that immunotherapies can be effective even in advanced cancer patients. Both antigen-specific (2,3) and nonantigen specific agents (2) have been approved by the Food and Drug Administration. Considering the amount of effort poured into the development of these novel agents, PET-based monitoring will advance our knowledge of the immunological processes that precede the failure or success of novel immunotherapies. Furthermore, it should vastly improve efficient application by aiding in individualized decision making. References 1. Hodi FS, et al. Improved survival with ipilimumab in patients with metastatic melanoma. N Engl J Med. 2010;363:711-723. 2. Schwartzentruber DJ, et al. gp100 peptide vaccine and interleukin-2 in patients with advanced melanoma. N Engl J Med. 2011;364:2119-2127. 3. Kantoff PW, et al. IMPACT Study Investigators Sipuleucel-T immunotherapy for castration-resistant prostate cancer. N Engl J Med. 2010;363:411-422.5 9. Lesterhuis WJ, et al. Dendritic cell vaccines in melanoma: From promise to proof? Crit Rev Oncol Hematol. 2008;66:118-134. 4. Banchereau J, Palucka AK. Dendritic cells as therapeutic vaccines against cancer. Nat Rev Immunol. 2005;5:296-306. 5. Shields AF, et al. Imaging proliferation in vivo with [F-18]FLT and positron emission tomography. Nat Med. 1998;4:1334-1336. 6. Troost EG, et al. 18F-FLT PET does not discriminate between reactive and metastatic lymph nodes in primary head and neck cancer patients. J Nucl Med. 2007;48:726-735. 7. Aarntzen EH, Srinivas M, De Wilt JH, Jacobs JF, Lesterhuis WJ, Windhorst AD, Troost EG, Bonenkamp JJ, van Rossum MM, Blokx WA, Mus RD, Boerman OC, Punt CJ, Figdor CG, Oyen WJ, de Vries IJ. Early identification of antigen-specific immune responses in vivo by [18F]-labeled 3′-fluoro-3′-deoxy-thymidine ([18F]FLT) PET imaging. Proc Natl Acad Sci U S A. 2011;8:108(45):18396-18399. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr SY24-02. doi:1538-7445.AM2012-SY24-02

We present evidence that human blood eosinophils produce interleukin (IL)-8 when stimulated with calcium ionophore. Following in vitro culture of 99 % pure eosinophils with calcium ionophore, released IL-8 was detectable by enzymelinked immunosorbent assay in supernatants. Eosinophil IL-8 production was considerably greater than that of IL-3 or granulocyte macrophage colony-stimulating factor. Furthermore, eosinophil production of IL-8 in the presence of calcium ionophore could be inhibited with the immunomodulating agent cyclosporin A and the protein synthesis inhibitor cycloheximide. In addition, following stimulation of highly purified blood eosinophils with calcium ionophore, IL-8 mRNA was detectable after polymerase chain reaction amplification. In comparison with other cells on stimulation with calcium ionophore, eosinophils produce about half as much IL-8 as neutrophils but significantly more than purified T cells. In contrast to monocytes and neutrophils, IL-8 production was not inducible with IL-1 or tumor necrosis factor. Finally, following calcium ionophore stimulation blood eosinophils were shown to contain cytoplasmic IL-8 by employing a monoclonal antibody against IL-8 in conjunction with immuno-histochemistry. These observations demonstrate that eosinophils are capable of IL-8 production and release, which may contribute to defense against parasites and to the pathophysiology of allergic and asthmatic disease.

It has become evident that many immune inhibitory mechanisms in the tumor micro-environment prevent the development of effective T cell responses against the tumor cells in cancer patients. Therapeutic interventions aimed at blocking these pathways have been shown to result in enhanced anti-tumor immunity, but they lack direct cytotoxic effects. Cancer chemotherapeutic drugs have these direct cytotoxic anti-tumor effects and although for some anti-cancer drugs indirect activation of the immune system via induction of an immunogenic type of tumor cell death has been described, their effect on inhibitory pathways in anti-tumor immunity is unknown. We investigated the effect of a wide array of chemotherapeutics on dendritic cell (DC) function and on tumor cell immunogenicity. Using allogeneic and antigen-specific in vitro models, we found that when DCs were matured/activated in the presence of platinum-based chemotherapy, they showed strongly enhanced T cell stimulatory capacity. Programmed death receptor-ligand 2 (PD-L2) expression by DC was markedly reduced upon platinum exposure. The enhanced T cell stimulatory capacity by DC upon platinum exposure was abrogated in the presence of PD-L2 blocking antibodies. This was also observed when the regulator of PD-L2 expression, signal transducer and activator of transcription 6 (Stat6), was knocked down using siRNA. In addition, we found that STAT6 was dephosphorylated in tumor cells by platinum compounds, leading to marked downregulation of PD-L2, resulting in enhanced recognition by tumor-specific T cells. In line with these in vitro findings, we found enhanced Th1 cytokine secretion and T cell proliferative responses in cancer patients upon treatment with platinum-based chemotherapy, confirming the in vivo occurrence of platinum-induced immune deviation. Our findings show that treatment with platinum compounds not only results in enhanced T cell stimulation by DC, but at the same time enhances the sensitivity of the tumor for lysis by cytotoxic T cells. This novel double action of platinum compounds may initiate combinations with other immune stimulatory compounds to increase clinical efficacy. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3823.

The Wilms’ tumor protein WT1 is a target for immunotherapy in malignancies, such as acute myeloid leukemia (AML). Following our demonstration that dendritic cells (DC) can be efficiently transfected by messenger (m)RNA electroporation (Van Tendeloo VF et al. Blood2001;98:49–56) and that WT1 mRNA-electroporated DC stimulate WT1-specific T cells in vitro (Van Driessche A et al. Leukemia2005;19:1863–1871), we performed a phase I/II dose-escalation trial, in which patients with AML in remission but at high risk of relapse and without a direct sib allo-transplant option (9 patients) or with slowly progressive AML (1 patient) received intradermal injections of WT1 RNA-loaded DC. Following apheresis and CD14 immunomagnetic monocyte separation, DC were generated in 6-day cultures in clinical-grade medium supplemented with serum, granulocytemacrophage colony-stimulating factor (GM-CSF) and interleukin (IL)-4, matured with prostaglandin (PG)E2 and tumor necrosis factor (TNF)-alpha, harvested, electroporated with WT1 mRNA and used as vaccines. The patients received four biweekly DC vaccines and a delayed-type hypersensitivity (DTH) test was performed 2 weeks following the last vaccination. Patients were monitored for minimal residual disease (MRD) by analyzing WT1 RNA expression in peripheral blood by quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) (Cilloni D et al. Leukemia2002;16:2115–2121 & Cilloni D et al. Haematologica2008;93:921–924). Before and after vaccination, peripheral blood was collected for immunomonitoring purposes. Feasibility, safety, immunogenicity and effect on MRD were investigated. There was successful DC generation and vaccine production in all 10 patients. No serious adverse events or toxicity were observed and vaccinations were well tolerated. A decrease in WT1 RNA expression was observed during the course of the vaccination in 4/7 patients who had an increased WT1 mRNA level in peripheral blood. Three of those patients are still in complete hematological remission. An in vivo vaccine-specific immune response was demonstrated in 10/10 patients by DTH. Ex vivo immunomonitoring analysis showed a significant increase in circulating activated HLA-DR+ CD4+ T cells and in IL-2 plasma levels following vaccination. Importantly, in vitro restimulation assays of peripheral blood mononuclear cells revealed a significant postvaccination increase in interferon (IFN)-gamma-producing WT1-specific CD8+ T cells (n= 8 evaluable patients), but not in cytokine-producing WT1-specific CD4+ T cells. There was no significant change in WT1-specific antibodies following vaccination. We conclude that vaccination of AML patients with WT1 RNA-loaded DC is feasible and safe. Furthermore, the DC elicit vaccine-specific and WT1-specific CD8+ T-cell responses. The correlation between reduction of circulating WT1 mRNA and the administration of the DC vaccines strongly suggests that this DC vaccine elicits an antileukemic activity.

Contains fulltext : 80991.pdf (Publisher’s version ) (Open Access) The occurrence of vitiligo in patients with melanoma is especially reported for patients undergoing immunotherapy. While vitiligo in these patients is thought to be related to an immune response directed against melanoma cells, solid evidence is lacking. Here we report local cytotoxic T cell reactivity in three melanoma patients who developed vitiligo, after experimental immunotherapy using dendritic cell vaccinations. Tetramer analysis showed that vaccine-induced T cells recognizing gp100 and tyrosinase are present at the vitiligo lesions. These T cells secrete IFN-gamma and IL-2 upon peptide specific stimulation as well as upon recognition of the autologous tumor. We show that functional CD8(+) T cells specific for melanoma differentiation antigens used in a melanoma immunotherapy trial, do not only invade the tumor, but also the vitiligo lesions. This directly links vitiligo to the immuno-therapeutic intervention and supports the hypothesis that vitiligo is a marker of immunity against melanoma cells.

To date, Wilms’ tumor protein (WT1) is acknowledged as a valuable target for active specific immunotherapy in several solid and hematological malignancies, such as leukemia. Preclinical data from our laboratory and that of Hans Stauss have already shown that WT1 RNA-electroporated dendritic cells (DC) stimulate WT1-specific T cells in vitro (Van Driessche A et al. Leukemia2005;19:1863–1871). Therefore, we started a phase I/II dose-escalation trial in which patients with acute myeloid leukemia (AML) in remission received intradermal injections with WT1 RNA-loaded DC. Feasibility, safety and immunogenicity of the vaccine were investigated. Seven patients received four biweekly DC vaccines. A delayed-type hypersensitivity (DTH) test was performed 2 weeks following the last vaccination. Patients underwent an apheresis and monocytes were isolated using CD14-labeled magnetic beads by CliniMACS. DC were generated in 6-day cultures in clinical-grade medium supplemented with serum, GM-CSF and IL-4 and maturated with PGE2 and TNF-a. Keyhole limpet hemocyanin (KLH) was added during maturation as a CD4+ helper antigen. Mature DC were harvested, electroporated with WT1 mRNA and used as vaccines. Patients were monitored for minimal residual disease (MRD) by analyzing WT1 RNA expression in peripheral blood by qRT-PCR. When the patient was HLA-A2+, tetramer staining was performed to detect WT1-specific CD8+ T cells. Before and after the vaccination cycle, peripheral blood was collected for immunomonitoring purposes. There was successful DC generation and vaccine production in all patients selected. No serious adverse events or toxicity was seen and all vaccinations were well tolerated. A decrease in WT1 RNA expression was observed during the course of the vaccination in 3/5 patients who had an increased WT1 mRNA level in peripheral blood at the start of DC vaccination. A vaccine-specific immune response was demonstrated in 7/7 patients by an in vivo DTH reaction both to KLH as well as to WT1. By tetramer analysis, detectable levels of WT1-specific CD8+ T cells could be demonstrated during the course of the vaccination both in the peripheral blood as well as in the expanded DTH-infiltrating T cells from the skin biopsies. Preliminary data from immunomonitoring in pre- and post-vaccination T cell samples from 3 patients show a mixed T helper (Th)1/Th2 response towards the KLH and the WT1 protein following vaccination. We conclude that vaccination of AML remission patients with WT1 RNA-loaded DC is feasible and safe. Furthermore, the vaccine elicits anti-vaccine T-cell responses in vivo and a decrease in WT1 RNA expression levels was observed during MRD monitoring in some vaccinated patients.

Abstract 16 Immunization using tumor antigen-loaded dendritic cells (DC) holds promise for the adjuvant treatment of cancer to control residual disease. In a phase I/II trial, we investigated the effect of autologous DC vaccination in 17 patients with acute myeloid leukemia (AML) in remission but at high risk of full relapse. Wilms’ tumor 1 protein (WT1), a nearly universal tumor antigen, was chosen as an immunotherapeutic target because of its established role in leukemogenesis and superior immunogenic characteristics. Two out of 3 patients, who were in partial remission with morphologically demonstrable disease after chemotherapy, were brought into complete remission following 4 biweekly intradermal injections of WT1 mRNA-electroporated DC. In those 2 patients as well as in 7 other patients who were in complete remission but who had molecularly demonstrable residual disease, there was a return to normal of the AML-associated WT1 mRNA tumor marker following DC vaccination, compatible with clinical and molecular response in 9/17 patients. In 3 patients, the WT1 mRNA tumor marker returned to pathological values following normalization after initial DC vaccination and additional injections of DC were needed to bring back the tumor marker to normal. Of the 9 responders, 3 have relapsed and 2 have died. Of the 8 non-responders, 7 have relapsed and 7 have died. Of the 2 patients in partial remission who were brought into complete remission by the DC vaccination, 1 has relapsed and has died. Median overall survival was 52.0 months in responders as compared to 6.0 months in non-responders (P=0.0007). Median relapse-free survival was 47.0 months in responders and 3.0 months in non-responders (P smaller than 0.0001). Immunomonitoring performed on the first 10 patients, showed a significant increase in WT1-specific interferon-gamma+ CD8+ T cells and signs of general immune stimulation, such as a significant increase of plasma levels of interleukin 2 and of HLA-DR+ CD4+ T-cells. Clinical responses were correlated with elevated levels of activated natural killer cells post-vaccination. Long-term clinical responses, lasting for at least 3 years, were significantly correlated with an increase in polyepitope WT1-specific tetramer+ CD8+ T-cell frequencies. There was no significant change post-vaccination in WT1 antibody levels or of regulatory T lymphocytes. In conclusion, DC-based immunotherapy elicits both innate and adaptive cellular immune responses correlated with clinical benefit. WT1 mRNA-loaded DC emerge as a feasible and effective strategy to control residual disease in AML, in particular as a post-remission treatment to prevent full relapse. Disclosures: Berneman: Argos Therapeutics: Patents & Royalties. Van Tendeloo:Argos Therapeutics: Patents & Royalties.