OR

Cells are subsequently separated into monocytes and lymphocytes by elutriation in a closed system (Elutra). The two most monocyte-rich fractions collected contained > 80% monocytes, the fraction richest in lymphocytes contained > 90% lymphocytes and viability in both fractions was > 95%.

Monocytes are isolated from PBMC using MACS anti-CD14 microbeads and a Midi-MACS® magnetic cell sorting device (both from Miltenyi Biotec, Bergisch- Gladbach,Germany).

Differentiation of monocytes into dendritic cells is promoted by addition of granulocyte-macrophage colony-stimulating factor (GMCSF 1000U/ml, Chemicon) and recombinant IL-4 (1000U/ml, R&D Systems) for 5 days.

After the centrifugation (10’ at 5000 rpm) the supernatant is transferred into a pre-spun (5’ at 1500 rpm) 50 ml Phase Lock Gel tube (Eppendorf 0032 005.330) and 4 ml of chloroform (Fischer BP1145-1) are added into the falcon.

Data are normalized to mean ratio intensity.

The monocytes are cultured for five days to immature DC (imDC´s).

Monitoring of immunological responses elicited by the DC vaccine and transferred T cells during the clinical trial

The patients are monitored for safety and toxicity of the treatment and for clinical response. T cell stimulatory activity of DC is monitored by induction of KLH specific T cells, tumor specific responses are evaluated by induction of SK-Mel 24 peptides specific T cells.

A HLA-A2 positive healthy donor undergoes leukapheresis.

Absorbance is measured using a microplate reader at 620 nm.

After 2 rounds of in vitro stimulation, the microcultures are left without stimulation for 2 weeks.

CD8 T cells are purified using MACS cell separation.

Patients will undergo lymphodepletion prior to vaccination and prior to adoptive transfer of autologous T cells.

Patients will undergo leukapheresis.

Leukapheresis will be performed on the following day.

HLA-A1 and/or HLA-A2 positive patients with stage III/IV melanoma undergo leukapheresis.

Patients receive V administrations with eventual additional ones depending on clinical outcome.

The next day the peptides and KLH are replaced and incubated for further 4h and then used for quality controls and intradermal administration.

PBMC are isolated from buffy coats by density gradient centrifugation using Biocoll (BIOCHROM).