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Homing of DC transfected with RNA encoding E/L-Selectin

signalling dataset

Data has been obtained on the homing capacity of DC transfected with RNA encoding E/L-Selectin.

We have efficiently transfected DC with RNA encoding a functional protein (E/L-Selectin) which allows entry of DC into LN from HEV. Mouse E/L-S transfected DC, when given i.v., homed to LN, whereas non transfected DC home only to the spleen. The novel adherence capacity of human E/L-S transfected DC was demonstrated via sticking to sialyl-LewisX coated slides using a parallel plate flow microscope. RNA transfected human DC could be frozen and thawed without loosing their functionality.

In an ongoing clinical trial (see WP9) using monocyte-derived and cocktail-matured DC electroporated with a combination of RNAs encoding tumor antigens (MelanA, Mage3, Survivin) and E/L-selectin, we already obtained superior immune responses using E/L-S+ DC compared to E/L-S- DC.

To demonstrate in-vivo that multiple LN regions can be targeted by i.v. application of E/L-S DC, we established indium111 labelling of DC and studied its effect on DC migration and T-cell interaction in vitro prior going into patients. In terms of migratory capacity, which was analyzed using a transwell assay adressing CCR7-mediated migration of DC towards MIP3-ß, we could not find any inhibition by labeling of DC with indium111 up to 4 MBq (100 µCi). In contrast, priming and expansion of MelanA specific naïve CD8 T-cells by DC loaded with MelanA analog peptide or MelanA encoding RNA, was markedly affected by indium111 labeling in a dose dependent manner. Whereas MelanA specific T cell expansion was only slightly reduced using DC labelled with 1MBq of indium111, 2MBq already completely abrogated specific T cell expansion and labelling with 4MBq even resulted in death of MelanA specific T cells. Therefore Indium111 labelled DC should not be loaded with tumor antigens if applied in patients, which may result in loss of specific immune responses at least at higher labelling intensities usually employed. Interestingly, this aspect had never been uncovered or considered in work done so far by other groups injecting Indium labelled DC previously.





created over 14 years ago (2 March 2009)    last modified over 11 years ago (16 July 2012)   [ RDF Rdf ]   [ RelFinder Relfinder ]