Cells from healthy blood donors are separated into monocytes (and lymphocytes) by elutriation in a closed system (Elutra). [The two most monocyte-rich fractions collected contained > 80% monocytes, viability was > 95%.]

After using frozen MoDC for tumor peptide specific stimulation of autologous T lymphocytes at a ratio of 1 DC per 10 T cells, co-cultures led to antigen specific, IFN-? secreting T cells (1 restimulation at a DC:T cell ratio of 1:20).

Cells are separated into lymphocytes (and monocytes) by elutriation in a closed system (Elutra). [The fraction richest in lymphocytes contained > 90% lymphocytes and viability was > 95%.]

These cells were stained by Mini67-1KT or Mini26-1KT (PKH67/PKH26 green/red fluorescent cell linker mini kit for general cell membrane labelling, from Sigma) in order to analyse their uptake of dead cells.

It was found that lymph nodes that drain sites of mature DC or adjuvant inoculation recruited memory CD8+ T cells.

Our current efforts in a study of functional homogeinity involve a transition to 8 colours surface staining, and the evaluation of antigen-specific cell sets identified with MHC tetramers. For the later process, we initiated data collection of EBV, CMV and HIV specific T cell sets.

Our current efforts in a study of functional homogeinity involve a transition to 8 colours surface staining, and the evaluation of antigen-specific cell sets identified with MHC tetramers. For the later process, we initiated data collection of EBV, CMV and HIV specific T cell sets.

It was found that lymph nodes that drain sites of mature DC or adjuvant inoculation recruited L-selectin- CCR7- effector cells.

It was found that these cells do not migrate to lymph nodes in the steady state.

Perforine expressing T cells were observed in human Hemato-Lymphoid System Rag2-/-gc-/- mice after being infected with EBV and mounting an immune response. They infiltrated in B cell infected areas in lymphoid organs in situ.

Granzyme expressing T cells were observed in human Hemato-Lymphoid System Rag2-/-gc-/- mice after being infected with EBV and mounting an immune response. They infiltrated in B cell infected areas in lymphoid organs in situ.

We also show that DCs derived from ashen mice, which are defective for the small GTPase Rab27a, fail to cross present antigens efficiently, due to increased phagosome acidification and antigen degradation. This defect in Rab27a-deficient DCs results from the impaired recruitment to phagosomes of the NOX2 membrane components. Therefore phagosomal alkalinization by NOX2 is controlled by Rab27a, and is required for efficient cross presentation in DCs.

Monocyte-derived and cocktail-matured DC electroporated with a combination of RNAs encoding tumor antigens (MelanA, Mage3, Survivin) and E/L-selectin are currently used in a clinical trial.

A comprehensive protein inventory of clinical grade immature and cytokine cocktail matured (Il-6, IL-1 beta, TNF alpha, PGE2; 48 hours) monocyte derived human dendritic cells (DC) from a healthy donor has been established by using high accuracy, high sensitivity protein identification technology. We have identified 2794 proteins in DCs by liquid chromatography tandem mass spectrometry. Prior to MS analysis, DC were lysed and divided into a soluble fraction containing cytosolic proteins and into an insoluble fraction enriched for membrane containing proteins. Hi...

We have stimulated human Monocyte derived Dendritic Cells (hMoDC) with yeast, spheroplasts, pseudohyphae, spores, purified RNA and not purified RNA, LPS.

These DC were used in an experiment for identification of antigens recognised by T cells from melanoma patients responding to immunotherapy.

RCC product that was tested in an RCC phase 2 trial. The product does indeed lead to restoration of both IL-2 and IFN responses and leads to a gradual decline of the immune effector T cells in the peripheral circulation (i.e., weaker signal after the fifth dose). Using the PME-CD40L DC product we have observed tumor regressions in a number of patients in the current study. At present, 6 of 12 patients that have been restaged using RECIST criteria have less tumor burden that when they enrolled in the study (15 weeks earlier).

For studying in vivo responses to antigens, we initiated a long program comparing the behaviour of TCR-Tg cells specific for GP-33 peptide.

Our current efforts in a study of functional homogeinity involve a transition to 8 colours surface staining, and the evaluation of antigen-specific cell sets identified with MHC tetramers. For the later process, we initiated data collection of EBV, CMV and HIV specific T cell sets.

Testing the improved RCC product PME-CD40L DC it is noteworthy that presence of CD28+ T cells in tumor lymphocytic infiltrates correlates with favorable clinical outcome.