After 10’ of incubation in 65 °C water bath, the samples were incubated on ice.

The yeast cells were resuspent in 3.6 ml AE Buffer and then transferred in 15 ml tubes wit 200 µl 10% SDS (w/v). 2 ml of preheated acid phenol (pH 4.3) were added and the falcon were mixed by vortex.

These autologous DC were used in a phase IB/II study of immunotherapy. Patients were randomized to receive immunizations either with I3 DC (DC generated with interferon-beta and interleukin-3) or with G4 DC (DC generated with GM-CSF and IL-4).

The T blasts are used to immunize MAGE-3 positive patients.

The autologous DC loaded with KLH, as immunological tracer, and an allogeneic peptidome (i.e. natural tumour peptides, NTPs), obtained from melanoma cell line SK-Mel24, are used for vaccination.

The autologous T lymphocytes were stimulated with (frozen) MoDC at a ratio of 1 DC per 10 T cells.

B cell proliferation (polyclonal, oligoclonal, monoclonal) was observed in human Hemato-Lymphoid System Rag2-/-gc-/- mice after being infected with EBV and mounting an immune response.

These T cells were generated from the Leukapheresis product, after elutration, of a metastatic lesion of stage III/IV melanoma patients, expanded by co-culture with tumor lysate loaded DC.

These mice were used in a DC staining experiment. We have started to visualize and characterize the DC which carry I-Ad/LACK complexes at the cell surface in BALB/c mice infected by leishmania major.

These not immunized mice were used in a DC staining experiment. To investigate whether several mAb could be used to detect APC presenting LACK in vivo, BALB/c mice were immunized or not with either LACK or OVA. LN cells were purified 2 days later, and stained with either 2C44, 2F74, 2E60 or 2X8 mAb. None of these mAb stained DC purified from non immunized mice. However, among the 4 mAb, only 2C44 could stain DC in LACK-immunized mice, but not in OVA-immunized mice. Furthermore, 2C44 stained DC from LACK-immunized mice expressing the H2-d haplotype but did no...

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

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

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

These mice were immunized with LACK and used in a DC staining experiment. LN cells were purified 2 days later, and stained with either 2C44, 2F74, 2E60 or 2X8 mAb. None of these mAb stained DC purified from non immunized mice. Among the 4 mAb, only 2C44 could stain DC in LACK-immunized mice, but not in OVA-immunized mice.

To address the issue whether infected DC process pathogen-derived antigen and stimulate antigen-specific CD4+ T cells in vivo, we have infected susceptible BALB/c (H2-d) mice with a recombinant Leishmania major parasite expressing a fluorescent tracer. We have directly visualized antigen-presenting cells using a mAb reacting to an antigenic peptide derived from the parasite LACK antigen bound to I-Ad MHC class II molecule. I-Ad/LACK complexes were readily detected at the surface of freshly purified parasite-containing DC which expressed a CD8?- CD11b+ F4/80+ g...

These dendritic cells were generated from adherent peripheral blood monocytes, subject to quality controls, and subsequently used for intradermal administration in HLA-A1 and HLA-A2 positive patients with stage III/IV melanoma (V administrations with eventual additional ones depending on clinical outcome).

The B-CLL patients were vaccinated with autologous, apoptotic, leukemic cells (Apo-DC). The first cohort of patients received the vaccine without additional adjuvants, while the next cohort of 5 patients who receive the vaccine with GM-CSF as an adjuvant. Clinical trial on vaccination of B-CLL patients with autologous, apoptotic, leukemic cells (Apo-DC), progressed further in 2007. Cohort 1 with the vaccine alone and cohort 2 (5 patients) combining the vaccine together with GM-CSF as an adjuvant has finished accrual and all patients in these two cohorts have ...

This colony of transgenic mice that over-express the rat HER-2/neu oncogene under the MMTV promoter were used to study new strategies of DC-based and other therapies for advanced breast cancer. Founders were provided by Forni, Torino IT.

We evaluated the capacity of autologous DC and HEK293 cells transfected with relevant HLA alleles to function as T cell targets in Elispot assays upon transfection with tumor antigen encoding plasmids. Due to strong background from the autologous DC we decided that HEK293 transfected with one HLA-allele at a time plus simultaneously transfected with up to 5 tumor antiges would be optimal to screen for antigen specificity in the patients T cells. However, during a second T cell culture to expand large numbers of T cells for this screening procedure a drastic ex...