TLR ligands (simultaneous activation of TLR4 and TLR8 signaling cascades) were used as stimuli for MDDC and resulted in a marked inhibition of the secretion of the proinflammatory chemokine CCL2 with respect to stimulation through a single TLR.

The autologous, apoptotic, leukemic cells (Apo-DC) vaccine was given to 2 cohorts of patients: without additional adjuvants and with GM-CSF as an adjuvant. These two cohorts were monitored for 52 weeks. The clinical and immune monitoring data is being currently analyzed.

CD1 molecules are refolded in vitro in order to generate CD1 tetramers.

CD1 molecules that are subsequently to be refolded in vitro in order to generate CD1 tetramers.

The protein vaccines were injected combined with a combination of iNKT cell-agonist alpha-GalCer and MPL, a detoxified version of LPS that signals through TLR4. This combination treatment stimulates antigen-specific T and B cell responses that are greater than those elicited with alpha-GalCer or MPL alone.

Differentiation of monocytes into dendritic cells is promoted, along with granulocyte-macrophage colony-stimulating factor, by addition of recombinant IL-4 (1000U/ml, R&D Systems).

Constructs for fusion proteins of the DC specific receptor DC-SIGN fused to GFP have been completed and tested.

100 micrograms/ml curdlan (Wako) was used (along with R848, LPS, yeast RNA and yeast cells in different conditions of culture) to induce DC activation.

LPS (1microgram/ml, Sigma, St. Louis, MO) (along with R848, Curdlan, yeast RNA and yeast cells in different conditions of culture) was used to induce DC activation.

Differentiation of monocytes into dendritic cells is promoted, along with recombinant IL-4, by addition of granulocyte-macrophage colony-stimulating factor (GMCSF 1000U/ml, Chemicon).

50 mg/ml of streptomycin (Gibco BRL) containing 10% (vol/vol) FCS (Hyclone) were added to the monocyte cell culture.

50 U/ml of penicillin were added to the monocyte cell culture.

100 mM sodium pyruvate were added to the monocyte cell culture.

1% (vol/vol) non-essential amino acids were added to the monocyte cell culture.

2 mM L-glutamine (Sigma) was added to the monocyte cell culture.

TNF-alpha is used during maturation of the DCs.

The vaccine is composed by autologous DCs electroporated with mRNA encoding CD40L, CD70, caTLR4 and one tumorantigen (gp100, Tyrosinase, Mage-C2 or Mage-A3).

The agonists were used as adjuvants in vivo to induce DC activation and CD4+ T cell and antibody responses.

This newly engineered antibody specific for alpha-galactosylceramide (alpha-GalCer)-human CD1d (hCD1d) complexes was used to measure the affinity of binding of iNKT TCR to hCD1d molecules loaded with a panel of alpha-GalCer analogues and to assess the rate of dissociation of alpha-GalCer and alpha-GalCer analogues from hCD1d molecules.

We analysed the ability of invariant NKT (iNKT) cells to assist priming of antigen specific T and B cell responses. 1) We have demonstrated that activation of human DC by Toll like receptor ligands (TLR-L) modulates the lipid biosynthetic pathway, resulting in enhanced recognition of CD1d-associated lipids by iNKT cells. 2) We have clarified the mechanisms by which CD1d-restricted lymphocytes translate T cell receptor (TCR) recognition of lipids with similar group heads into distinct biological responses. Using a soluble iNKT TCR and a newly engineered anti...