B16TRex cell line cells were transfected with Fadd-dd to induce necrosis.

The B16TRex cell line cells were transfected with Bims to induce apoptosis.

The immature monocyte-derived dendritic cells where transduced with high doses of lentiviral vectors to monitor activation of the immature DC.

Autologous DC were pulsed with apoptotic allogenic prostate carcinoma cell line LNCap to produce the advanced prostate carcinoma vaccine.

Alternatively activated DC (AA-DC) were obtained by culturing immature DC in the presence of maturative agonists (such as, LPS, CD40L or TNF) and calcitriol, IL-10 or prostaglandin E2 for a study of molecular signatures for alternatively activated DC. AA-DC showed a decrease in the production of IL-12 but retained most of the ability to secrete other cytokines, such as TNF and CXCL8. The hallmark of AA-DC was the production of the angiogenic cytokine VEGF in vitro, and in vivo when the cells were implanted into the chicken embryo CAM assay.

These autologous DC were electroporated with mRNA encoding CD40L, CD70, caTLR4 and one tumorantigen (gp100, Tyrosinase, Mage-C2 or Mage-A3) to produce a vaccine for immunotherapy of advanced melanoma patients.

These carcinoma cells were used to pulse autologous DCs for vaccine production for ovarian carcinoma patients.

These autologous dendritic cells were used for vaccine production. They were pulsed with apoptotic autologous ovarian carcinoma cells.

The properties of this DC population and its production of cytokines in response to different Toll-like receptor (TLR) agonists were evaluated. The TLR agonists used were: PAM3CSK4, Poly I:C, LPS, Flagellin, Imiquimod, Resiquimod, CpG 2216, CpG 2006. We found that CD16 and CD1c produce a number of cytokines in response to these stimuli, with the exception of CpGs. In addition, we found that CD16+ DCs are the major producers of TNF-alpha and IL-6, while CD1c+ DCs produce primarily IL-8.

The properties of this DC population and its production of cytokines in response to different Toll-like receptor (TLR) agonists were evaluated. The TLR agonists used were: PAM3CSK4, Poly I:C, LPS, Flagellin, Imiquimod, Resiquimod, CpG 2216, CpG 2006. We found that CD16 and CD1c produce a number of cytokines in response to these stimuli, with the exception of CpGs. In addition, we found that CD16+ DCs are the major producers of TNF-alpha and IL-6, while CD1c+ DCs produce primarily IL-8.

The properties of this DC population and its production of cytokines in response to different Toll-like receptor (TLR) agonists were evaluated. The TLR agonists used were: PAM3CSK4, Poly I:C, LPS, Flagellin, Imiquimod, Resiquimod, CpG 2216, CpG 2006. BDCA-3+ cells seem not have any capability to respond to TLR agonists.

We performed co-cultures of IFN-DCs with zoledronate-stimulated gamma delta T lymphocytes. Gamma delta T-cell activation, as demonstrated by their up-modulation of activation marker expression levels (e.g. CD25 and CD69), was observed. The studies demonstrated for the first time the existence of a bidirectional activating interaction between DCs and gamma delta T lymphocytes activated by aminobiphosphonates.

1,25(OH)2D3 was added to the freshly isolated monocytes; this prevented the generation of IFN-DCs and directed already differentiated IFN-DCs toward a more immature stage.

These DC were matured with inflammatory cytokines and subsequently electroporated with mRNA encoding 6 tumor-associated antigens. They were used for DC-based immmunotherapy of melanoma patients.

The kinetic of the transcriptional profiling of D1 cells treated with either DMSO or LPS was analysed.

Immature DC (imDC´s) are transfected with different constructs encoding for the oncogene Her-2/neu and as control PSA. The technology of Amaxa biosystems or an adeno virus Her-2 full length construct are used.

We identified a novel DC subset called IKDC producing IFNg and expressing TRAIL which can recognize and kill transformed tumor cells in vitro and in vivo. Cultures were generated of tumor cells with IKDC producing IFNg and expressing TRAIL which can recognize and kill transformed tumor cells in vitro and in vivo. IKDC generate long contacts and synapses with tumor cells and ultimately kill them within 4 hours.

HPV-18 E6 specific CD4+ T cells were found in a high percentage of patients with cervical lesions and we showed that the quality of the response (i.e. the level of IFN-? produced) could predict the clinical outcome after surgery.

MAGE-A3 specific CD4+ T cells were found also in a high percentage of melanoma patients but CD4+ T cells showed an unpolarized or Th2 skewed phenotype. We have optimized the MAGE-A3 peptides to load onto HLA-DR*1101 tetramers.

CEA specific CD4+ T cells were found both in normal donors and in patients with high-grade cervical lesions, pancreas adenocarcinoma and advanced melanoma but CD4+ T cells from the patients compared to normal donors showed impaired IFN-gamma production.