FRET microscopy autologous tumor lysate processing in mature dendritic cell vaccine therapy

Abstract

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.

Figure 1

Experimental plan and fluorescence images. (A) Scheme of the experimental plan. (B) SDS-PAGE analysis of ATL. Proteins gel electrophoresis separation was run on acrylamide gradient (4%-20%). The ATL sample is shown after Coomassie brilliant Blue staining of the proteins bands (lane 1) and by UV transillumination, before staining of the proteins bands, to reveal the extent of the fluorescence labelling (lane 2). (C) FRET analysis of mDC loaded with Alexa488-labeled ATL and immunolabelled with HLA-DR(HL12) mAb and Alexa546-conjugated IgG. The upper panels refer to the sample analyzed at 16 hours after the maturation stimulus and the lower panels refer to the sample analyzed at 46 hours after the maturation stimulus. Panels are divided in sets of images acquired before and after acceptor photobleaching (see Materials and Methods). Donor images were acquired in the green channel (a, h, d and k) and acceptor images were acquired in the red channel (b, i, e and l). White arrows (e and l) indicate the bleached regions. The relative merged images are also shown (c, j, f and m). FRET efficiency was calculated using eq. 1 and the results are presented as pseudo-color images (g and n).

Figure 2

FRET measurements. (A) Averaged FRET efficiency of mDC as a function of time from the maturation stimulus. The data and the Standard Errors (±SE) refer to FRET measurements performed over at least three fields for each sample (n = 3-5) and different ROIs (n = 30-55) inside the bleached regions. The x axis displays the time in culture after maturation stimulus. (B) Plot of the independence of E% from acceptor levels. The data shown were generated from image measurements 22 hr after maturation. The acceptor levels refer to the intensity of the image acquired before acceptor photobleaching and analyzed versus the recovered E%, on a pixel by pixel basis.