BackgroundActive dendritic cell (DC) immunization protocols are rapidly gaining interest as therapeutic options in patients with acute myeloid leukemia (AML). Here we present for the first time a GMP-compliant 3-day protocol for generation of monocyte-derived DCs using different synthetic Toll-like receptor (TLR) agonists in intensively pretreated patients with AML.MethodsFour different maturation cocktails were compared for their impact on cell recovery, phenotype, cytokine secretion, migration, and lymphocyte activation in 20 AML patients and 25 healthy controls.ResultsMaturation cocktails containing the TLR7/8 agonists R848 or CL075, with and without the addition of the TLR3 agonist poly(I:C), induced DCs that had a positive costimulatory profile, secreted high levels of IL-12(p70), showed chemotaxis to CCR7 ligands, had the ability to activate NK cells, and efficiently stimulated antigen-specific CD8+ T cells.ConclusionsOur results demonstrate that this approach translates into biologically improved DCs, not only in healthy controls but also in AML patients. This data supports the clinical application of TLR-matured DCs in patients with AML for activation of innate and adaptive immune responses.

2195 Introduction: Therapeutic vaccination with dendritic cells (DC) is currently considered as an investigational therapy in acute myeloid leukemia (AML) for eradication of minimal residual disease (MRD). Dendritic cells derived from autologous peripheral blood monocytes have been tested as cellular adjuvants for therapeutic vaccination of malignancies and proven feasibility and safety, but overall clinical response rates remain very low. The vast majority of DCs used for clinical trials were differentiated with a standard maturation cocktail composed of the cytokines TNF-a, IL-1b, IL-6 and PGE2 leading to DCs unable to secrete biologically active IL-12. This cytokine is fervently desired because of its leading role in promoting T helper 1 cell polarization and therefore fostering the appropriate adaptive immune responses needed to combat minimal residual disease. Cocktails containing synthetic Toll-like receptors (TLR) agonists emerged as an attractive alternative for the induction of DC maturation with T helper type 1 polarizing capacity. Our present investigation was designed to study the feasability of a clinical grade DC 3-day mDC generation protocol from nonleukemic monocytes of intensively pretreated AML patients with novel maturation cocktails containing different TLR-agonists in vitro and assessment of their potency to induce adaptive and innate immune responses. Material & Methods: Monocytes isolated from peripheral blood of AML patients in CR and healthy donors were differentiated into immature DC with GM-CSF and IL-4. After 48 hours DC were additionally cultured with TNF-a, IL1-b, INF-g, PGE2 and corresponding to the defined cocktail with the TLR7/8 agonists R848 (R) or CL075 (C) with or without the TLR3 agonist poly(I:C) (P) for 24 hours. mDCs were analyzed for expression of maturation surface markers, costimulatory profile, IL-12(p70)/IL-10 ratio, migratory capacity, NK cell activation and polarization of T cells. Results: No significant difference in absolute monocyte counts and percentage of DC recovery between healthy controls and AML patients in CR was found using different maturation cocktails (C, CP, R, and RP). Phenotype analysis of surface marker expression revealed no substantial differences between the different DC generation protocols used in healthy donors and AML patients in CR. The costimulatory profile assesed by the expression of two members of the B7 family, CD80 (B7.1) and CD274 (B7-H1 or PD-L1), was in healthy donors superior to AML patients, but these differences were not statistically significant. Variations were noted in the capacity of DCs derived from different donors to produce IL-12(p70) and IL-10, but importantly no significant differences between AML patients in CR and healthy controls could be observed. Interestingly, both healthy donor and AML derived DCs secrete a significantly higher proportion of IL-12(p70) with R848 containing cocktails compared to CL075. Treatment with the CP cocktails even leads to a inverse Il12/IL-10 ratio in AML patients. The high CCR7 expression was paralleled by a strong migratory capacity as well as positive chemotactic reponses to CCL19 chemokine signals. DCs matured with these novel cocktails induced potent alloresponses and strongly activated NK cells measured by upregulation of CD69 expression and IFN-g secretion. No differences beetween R848 and CL075 could be observed. Conclusion: Here we report for the first time a clinically applicable, time- and resource saving 3-day TLR-agonist containing maturation protocol for the generation of IL-12(p70) secreting DCs from AML patients in remission validated with healthy controls which allowed efficient generation, easy harvesting, stable maturation and substantial recoveries of mature DCs. Comparison of different TLR7/8 agonists showed superiority of R848 in IL-12(p70) production to CL075. We believe that these studies point the way to improved DCs that will induce better and long lasting immune responses in the vaccination against acute myelo Disclosures: No relevant conflicts of interest to declare.

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Fallschilderung Anamnese. Eine 63-jährige Patientin wird Ihnen mit anhaltendem Husten und leichten Hämoptysen vom Hausarzt zugewiesen. Es bestehen zudem eine Gewichtsabnahme von 4 kg in den letzten 4 Wochen und eine allgemeine Abgeschlagenheit. Die Patientin hat nie geraucht, Familien-und Berufsanamnese sind bland, Vorerkrankungen sind keine bekannt. Körperlicher Untersuchungsbefund. 63-jährige Patientin in gering reduziertem Allgemeinzustand (Eastern-Cooperative-Oncology-Group[ECOG]-Performance-Status 0-1) und schlankem Ernährungszustand (Größe 168 cm, Gewicht 63 kg). -Vitalparameter: Herzfrequenz 100/min, Blutdruck 130/90 mm Hg, Atemfrequenz 20/min, Sauerstoffsättigung (SO2) 96 %, Temperatur 37,3 °C -Haut und Schleimhäute unauffällig, Lymphknoten nicht vergrößert palpabel -Cor: tachykarde, reine, rhythmische Herztöne -Pulmo: ubiquitär vesikuläres Atemgeräusch -Abdomen: kein Druckschmerz, keine Abwehrspannung, regelrechte Darmgeräusche über allen Quadranten Auffällige Laborwerte (optional).-Leukozyten 11.000/μl (Referenzbereich 4000-10.000/μl) -Hämoglobin 13,8 g/dl (Referenzbereich 14-18 g/dl) -C-reaktives Protein 20 mg/l (Referenzbereich < 3 mg/l

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Angesichts der Tatsache, dass die Akupunktur in Deutschland, aber auch in anderen Ländern wie etwa den USA, zunehmend im Rahmen des sozialversicherungsfinanzierten Gesundheitswesens angeboten wird – und die Kosten übernommen werden –, stellt sich die grundsätzliche Frage, ob es sich (noch) um eine komplementäre Therapie in dem Sinne handelt, dass sie an dieser Stelle Gegenstand einer intensiveren Betrachtung sein sollte. Wie den Kommentaren zu den beiden ausgewählten Publikationen bzw. den dort zitierten Referenzen zu entnehmen ist, scheint die Akupunktur eine bestechend gute Publicity, aber nicht unbedingt eine vergleichbar überzeugende Evidence zu haben. Auch neuere Studien begeben sich offensichtlich methodisch aufs Glatteis und versuchen, den zweiten Schritt vor dem ersten zu tun. Andere scheinen zumindest zu bestätigen, dass die Akupunktur im besten Sinne komplementär (also ergänzend) wirksam ist. Der zweite Schwerpunkt gilt diesmal Aspekten der Ernährung/Diätietik. «Kann man Gesundheit essen?» mag sich der eine oder andere fragen, wenn er ratlos vor dem Joghurt-Regal steht. Es scheint, man kann, wenngleich trotz der plausiblen Ansätze vieles offensichtlich nicht endgültig experimentell geklärt ist. Anders als beim Thema Makrobiotik, für das gewichtige Gründe vorgebracht werden, warum diese Form der Ernährung (vor allem Kindern) ohne entsprechende Ergänzungen möglicherweise mehr schadet als nützt.