Prostaglandin E2 in a TLR3- and 7/8-agonist-based DC maturation cocktail generates mature, cytokine-producing, migratory DCs but impairs antigen cross-presentation to CD8+ T cells.

Mature dendritic cells (DCs) represent cellular adjuvants for optimal antigen presentation in cancer vaccines. Recently, a combination of prostaglandin E2 (PGE2) with Toll-like receptor agonists (TLR-P) was proposed as a new standard to generate superior cytokine-producing DCs with high migratory capacity. Here, we compare TLR-P DCs with conventional DCs matured only with the proinflammatory cytokines TNFα and IL-1ß (CDCs), focussing on the interaction of resulting DCs with CD8+ T-cells. TLR-P matured DCs showed elevated expression of activation markers such as CD80 and CD83 compared to CDCs, together with a significantly higher migration capacity. Secretion of IL-6, IL-8, IL-10, and IL-12 was highest after 16 h in TLR-P DCs, and only TLR-P DCs secreted active IL-12p70. TLR-P DCs as well as CDCs successfully primed multifunctional CD8+ T-cells from naïve precursors specific for the peptide antigens Melan-A, NLGN4X, and PTP with comparable priming efficacy and T-cell receptor avidity. CD8+ T-cells primed by TLR-P DCs showed significantly elevated expression of the integrin VLA-4 and a trend for higher T-cell numbers after expansion. In contrast, TLR-P DCs displayed a substantially reduced capability to cross-present CMVpp65 protein antigen to pp65-specific T cells, an effect that was dose-dependent on PGE2 during DC maturation and reproducible with several responder T-cell lines. In conclusion, TLR-P matured DCs might be optimal presenters of antigens not requiring processing such as short peptides. However, PGE2 seems less favorable for maturation of DCs intended to process and cross-present more complex vaccine antigens such as lysates, proteins or long peptides.

High-grade glioma associated immunosuppression does not prevent immune responses induced by therapeutic vaccines in combination with Treg depletion.

High-grade gliomas (HGG) exert systemic immunosuppression, which is of particular importance as immunotherapeutic strategies such as therapeutic vaccines are increasingly used to treat HGGs. In a first cohort of 61 HGG patients we evaluated a panel of 30 hematological and 34 plasma biomarkers. Then, we investigated in a second cohort of 11 relapsed HGG patients receiving immunomodulation with metronomic cyclophosphamide upfront to a DC-based vaccine whether immune abnormalities persisted and whether they hampered induction of IFNγ+ T-cell responses. HGG patients from the first cohort showed increased numbers of leukocytes, neutrophils and MDSCs and in parallel reduced numbers of CD4+/CD8+ T-cells, plasmacytoid and conventional DC2s. MDSCs and T-cell alterations were more profound in WHO IV° glioma patients. Moreover, levels of MDSCs and epidermal growth factor were negatively associated with survival. Serum levels of IL-2, IL-4, IL-5 and IL-10 were altered in HGG patients, however, without any impact on clinical outcome. In the immunotherapy cohort, 6-month overall survival was 100%. Metronomic cyclophosphamide led to > 40% reduction of regulatory T cells (Treg). In parallel to Treg-depletion, MDSCs and DC subsets became indistinguishable from healthy controls, whereas T-lymphopenia persisted. Despite low T-cells, IFNγ-responses could be induced in 9/10 analyzed cases. Importantly, frequency of CD8+VLA-4+ T-cells with CNS-homing properties, but not of CD4+ VLA-4+ T-cells, increased during vaccination. Our study identifies several features of systemic immunosuppression in HGGs. Metronomic cyclophosphamide in combination with an active immunization alleviates the latter and the combined treatment allows induction of a high rate of anti-glioma immune responses.

Immunotherapy in atypical teratoid-rhabdoid tumors: Data from a survey of the HGG-Immuno Group.

BACKGROUND AIMS: Atypical rhabdoid/teratoid tumors (AT/RT) are the most common brain tumors in infants and associated with a dismal prognosis. Although intensification of first-line therapy has resulted in improvement of overall survival, novel treatment strategies are needed. Because immunotherapy has resulted in remarkable results in several adult tumor entities, incorporation of immunotherapy into AT/RT treatment offers a novel alternative. METHODS: We retrospectively analyzed data from 7 AT/RT patients from five countries treated within the HGG-Immuno Consortium. Two patients were ≤1 year and 4 patients were ≤2 years of age at diagnosis. All received immunotherapy with autologous, tumor-lysate-loaded dendritic cells (DCs) on a compassionate use basis using a schedule of three to four weekly DC vaccinations with up to 2 × 10(7) DCs per vaccine, followed by three lysate boosts each 1 month apart. RESULTS: Monocyte collections (median age at apheresis 31.5, range 20-143 months) and vaccinations were uneventful without any severe adverse event related to the vaccine, demonstrating feasibility and safety in this very young age group. Two children received immunotherapy during their primary and the remaining five during second- or third-line therapy. Three of seven patients survived long term with a follow-up of 143, 138 and 46 months, with at least two of them harboring somatic mutations. One long-term survivor was vaccinated during primary treatment and the other two after first or second relapse/progression. Two analyzed patients showed positive CD8(+) T-cell responses after vaccination. DISCUSSION: Our data demonstrate that anti-tumor immunotherapy with autologous DCs is feasible and safe in young children with ATRTs and that this approach warrants further investigation in controlled clinical trials.

Development and validation of a fully GMP-compliant production process of autologous, tumor-lysate-pulsed dendritic cells.

BACKGROUND AND AIMS: One of the major challenges of dendritic cell (DC) vaccination is the establishment of harmonized DC production protocols. Here, we report the transfer and validation of a successfully used open DC manufacturing method into a closed system, good manufacturing practice (GMP)-compatible protocol. METHODS: All production steps (lysate generation, monocyte selection, DC culture and cryopreservation) were standardized and validated. RESULTS: Tumor lysate was characterized by histology, mechanically homogenized and avitalized. This preparation yielded a median of 58 ± 21 µg protein per milligram of tumor tissue. Avitality was determined by trypan blue staining and confirmed in an adenosine triphosphate release assay. Patient monocytes were isolated by elutriation or CD14 selection, which yielded equivalent results. DCs were subsequently differentiated in Teflon bags for an optimum of 7 days in CellGro medium supplemented with interleukin (IL)-4 and granulocyte macrophage colony stimulating factor and then matured for 48 h in tumor necrosis factor-α and IL-1ß after pulsing with tumor lysate. This protocol resulted in robust and reproducible upregulation of DC maturation markers such as cluster of differentiation (CD)80, CD83, CD86, human leukocyte antigen-DR and DC-SIGN. Functionality of these DCs was shown by directed migration toward C-C motif chemokine ligand 19/21, positive T-cell stimulatory capacity and the ability to prime antigen-specific T cells from naive CD8(+) T cells. Phenotype stability, vitality and functionality of DCs after cryopreservation, thawing and washing showed no significant loss of function. Comparison of clinical data from 146 patients having received vaccinations with plate-adherence versus GMP-grade DCs showed no inferiority of the latter. CONCLUSIONS: Our robust, validated and approved protocol for DC manufacturing forms the basis for a harmonized procedure to produce cancer vaccines, which paves the way for larger multi-center clinical trials.

Stimulating surface molecules, Th1-polarizing cytokines, proven trafficking--a new protocol for the generation of clinical-grade dendritic cells.

BACKGROUND AIMS: Dendritic cells (DC) have been vigorously investigated as an immunological basis for therapeutic vaccination against cancer and infections, even among patients after allogeneic stem cell transplantation. METHODS: Effective induction of cell-mediated immunity strongly depends on the ability of DC to (i) migrate to the draining lymphoid organs mediated by chemokine receptors, (ii) prime T cells through high expression of costimulatory molecules and major histocompatibility complexes and (iii) secret Th1-polarizing cytokines such as Interleukin-12 (IL-12). However, there is no protocol to generate fully matured and functional DC according to methodical requirements of current good manufacturing practice (CGMP) guidelines. RESULTS: We established a protocol conforming to CGMP standards that permits the generation of fully matured and functional DC on the basis of cell culture in adherence bags with the use of serum-free media with a maturation cocktail, containing tumor necrosis factor-alpha/Interferon-alpha/polyinosinic:polycytidylic acid. Our DC superiorly display three critical features for an effective induction of cell-mediated immunity without evidence of exhaustion, along with its ability to prime infectious or tumor-specific T cells in a short-term cell culture. CONCLUSIONS: Our newly developed protocol offers an attractive method to produce fully matured Th1-polarizing DC with proven migratory and stimulatory capacity for any clinical application according to CGMP standards.

Dendritic cell vaccination in pediatric gliomas: lessons learnt and future perspectives.

Immunotherapy of malignant gliomas with autologous dendritic cells (DCs) in addition to surgery and radiochemotherapy has been a focus of intense research during the past decade. Since both children and adults are affected by this highly aggressive brain tumor, 10-15% of the several hundred vaccinated patients represent children, making pediatric glioma patients the largest uniform pediatric vaccination cohort so far. In general, DC vaccination in malignant gliomas has been shown to be safe and several studies with a non-vaccinated control group could clearly demonstrate a survival benefit for the vaccinated patients. Interestingly, children and adolescents below 21 years of age seem to benefit even more than adult patients. This review summarizes the findings of the 25 clinical trials published so far and gives a perspective how DC vaccination could be implemented as part of multimodal therapeutic strategies in the near future.