PhD SCIENCE REPORT STEVE VOLAND: Immunomonitoring in stage IV melanoma patients vaccinated with dendritic cells

STEVE VOLAND

Dep. Dermatology, University Hospital Erlangen
Expected completion: January 2010

To improve the routine monitoring of tumour-specific T-cell subsets by multicolour flow cytometry, we established a protocol for the combination of multimer staining and intracellular cytokine staining. After antigenic stimulation, human cytolytic T lymphocyte (CTL) exhibit a decrease in their binding to human leukocyte antigen (HLA)-peptide tetramers, since CTLs lose the colocalization of the T cell receptor (TCR) with CD8. It has been suggested by the group of Pierre van der Bruggen (Brussels) that the TCR-CD8 colocalization may be restored using galectin disaccharide ligands.

We therefore investigated the effect of the galectin disaccharide ligand: N-acetyllactosamine (LacNAc) with regard to the loss of tetramer staining after antigenic stimulation and intracellular cytokine staining at the same time. Cells from a cytolytic T cell line specific for MelanA.A2 were stimulated for 4 hours with the peptide (10µg/ml) in the absence or presence of LacNAc. Afterwards the cells were washed and incubated with tetramer for 30 min at 25°C in the dark. Cells were then incubated for 15 min at 4°C with cell surface marker antigens (CD3/CD8) and washed. For intracellular staining cells were fixed with 1% formaldehyde, permeabilized with Saponin (Sigma) and stained for 15 min at 4°C with a FITC labelled mAb to interferon gamma. Samples were analysed on a FACS Canto II system.

We observed that cells which were not treated with LacNAc showed a secretion of interferon gamma, but lost the surface marker CD8 and tetramer labelling. Treatment with LacNAc nearly completely recovered the tetramer/CD8 staining if compared to unstimulated cells. LacNAc at 1 mM concentration did not hamper T cell activation, since tetramer positive cells showed an intracellular interferon gamma staining comparable to stimulated cells in the absence of LacNAc.

In summary the presence of the galectin disaccharide ligand: LacNAc during stimulation of the MelanA.A2 specific cytolytic T cells prevented surface loss of CD8 and dissociation between TCR and CD8, enabling simultaneous staining for tetramer and intracellular cytokines.

In addition, to determine the killing functionality of vaccination induced T-cells of patients in the CD40L multipeptide trial, we also started with chromium release assays after MLPC-stimulation. With this approach we monitor the lytic efficiency of T-cells against different target cells (T2A1 cells or autologous B-lymphoblastoide cells (B-LCL) loaded with tumor associated antigens (MAGE1.A1, MAGE3.A1, Tyrosinase.A1, NY-ESO1.A2, MAGE10.A2, Gp100.A2) or autologous tumor cells. Primarily results demonstrated the ability of stimulated T-cells to recognize and lyse peptide loaded T2A1 cells and in some patients also peptide loaded B-LCL and autologous tumor cell lines.

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