FRET microscopy autologous tumor lysate processing in mature dendritic cell vaccine therapy.
BACKGROUND
Antigen processing by dendritic cells (DC) exposed to specific stimuli has been well characterized in biological studies. Nonetheless, the question of whether autologous whole tumor lysates (as used in clinical trials) are similarly processed by these cells has not yet been resolved.


METHODS
In this study, we examined the transfer of peptides from whole tumor lysates to major histocompatibility complex class II molecules (MHC II) in mature dendritic cells (mDC) from a patient with advanced melanoma. Tumor antigenic peptides-MHC II proximity was revealed by Förster Resonance Energy Transfer (FRET) measurements, which effectively extends the application of fluorescence microscopy to the molecular level (<100A). Tumor lysates were labelled with Alexa-488, as the donor, and mDC MHC II HLA-DR molecules were labelled with Alexa-546-conjugated IgG, as the acceptor.


RESULTS
We detected significant energy transfer between donor and acceptor-labelled antibodies against HLA-DR at the membrane surface of mDC. FRET data indicated that fluorescent peptide-loaded MHC II molecules start to accumulate on mDC membranes at 16 hr from the maturation stimulus, steeply increasing at 22 hr with sustained higher FRET detected up to 46 hr.


CONCLUSIONS
The results obtained imply that the patient mDC correctly processed the tumor specific antigens and their display on the mDC surface may be effective for several days. These observations support the rationale for immunogenic efficacy of autologous tumor lysates.
Immunotherapy and Somatic Cell Therapy Laboratory, Istituto Scientifico Romagnolo per lo Studio e la Cura dei Tumori (IRST) Meldola, Italy.
Laura
L
Fiammenghi
Valentina
V
Ancarani
Tilman
T
Rosales
Jay R
JR
Knutson
Massimiliano
M
Petrini
Anna Maria
AM
Granato
Elena
E
Pancisi
Laura
L
Ridolfi
Ruggero
R
Ridolfi
Angela
A
Riccobon
Paolo
P
Neyroz
D019496:Cancer Vaccines / Q000276:immunology*; D002454:Cell Differentiation / Q000276:immunology; D003713:Dendritic Cells / Q000166:cytology / Q000276:immunology*; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D005455:Fluorescent Antibody Technique; D006801:Humans; D016233:Immunotherapy, Active / Q000379:methods*; D018613:Microscopy, Confocal / Q000379:methods*; D009369:Neoplasms / Q000276:immunology* / Q000473:pathology; D013997:Time Factors; D014020:Tissue Extracts / Q000276:immunology*
D019496:Cancer Vaccines; D014020:Tissue Extracts