Proof of the specific activation of immune responses is crucial in the overall rationale of cancer immunotherapy, and, more specifically, it is needed to convincingly address any analysis of immunogenic efficacy. In the present work we evaluated the presentation of ATL peptides onto MHC II of mDC from a patient with advanced melanoma.
The pattern of the protein content of ATL has been presented in Figure 1B together with the result of its homogeneous fluorescence labeling. These products were used previously to monitor the uptake and the processing of ATL in DC by fluorescence microscopy imaging . Here, the antigens (ATL) and antigen-capturing molecules (MHC II) were tagged to act as donor-acceptor pairs, and FRET measurements were performed to resolve the physical interactions between ATL and MHC II. Under these conditions, our results indicate a significant correlation between FRET efficiency and the time after maturation stimulus (Figure 2A). This observation is consistent with an increasing transfer of ATL peptide-loaded MHC II molecules on the mDC membrane. This process is significant 22 hr after maturation, and antigen presentation remains fully effective after 46 hr. The kinetic response observed is in excellent agreement with those reported on the transport of specific HEL-peptide-MHC II complexes at the DC surface , and the accumulation of MHC II complexes on mDC induced by inflammatory stimuli . Yet, in accord with these reports, the apparent discrepancy between the high levels of acceptor fluorescence and the absence of FRET detection shortly after maturation (≤4 hours), could possibly be related to the rapid turnover of unloaded MHC II molecules observed in developing DCs.
In Figure 2B we addressed the potential effects of MHC II density over FRET by plotting acceptor levels versus the efficiency, E%. This test was developed to study the distribution of proteins at the apical surface of MDCK cells . In particular, in the appendix of that survey, the theoretical dependence of FRET was separated into random or clustered distribution of donor- and acceptor-labeled molecules. It was clearly shown that the clustered model predicts that the efficiency will be independent of the surface densities of the labeled molecules. As mentioned above, given that newly synthesized class II molecules are produced in increased amounts in the first 24 hours after maturation , in our study we were particularly cautious about the FRET detection bias due to acceptor overcrowding . In this respect, a more distinctive feature of MHC II organization on the plasma membrane of DC was elucidated recently by Unternaehrer and coworkers  in which MHC II molecules were found to cluster by a lateral association mediated mechanism.
In our study, the independence of E% from acceptor levels (random distribution of E%) clearly indicates the absence of a nonspecific density contribution to FRET and fits the clustered model. Thus, we assign the significant increase of FRET efficiency, observed at the membrane surface as a function of time from maturation, to the actual transfer of specific tumor antigen peptides into MHC II clustered complexes.
This single-case survey on an advanced melanoma vaccination trial shows that autologous tumor lysates are correctly processed and presented at the mDC membrane surface in melanoma patients. In addition, this time-dependent profile is consistent with a delayed mDC antigen display, a property that is crucial for their role in vaccination-triggered immune surveillance . Yet, the methodology described and the parameters obtained (i.e. FRET signals) can be applied to follow-up studies to analyze and evaluate their prognosis value in addressing the efficacy of immunotherapy protocols.
Finally, it is worth commenting on the potential wealth of information that could be gleaned from FRET measurements when maximal FRET efficiency is known. In favorable circumstances, a quantitative data analysis approach is possible (i.e. a measure of the absolute changes in the amounts of antigen-loaded MHC II molecules at the DC membrane surface). Unfortunately, this information can only be obtained from extensive studies where appropriate standards are available (i.e. oligonucleic acid hybrids, streptavidin-biotin coupled donor-acceptor pairs) , or when specific tagged molecules can be engineered . Under our particular experimental conditions, we could not define the maximal FRET efficiency of the investigated donor-acceptor system (Alexa488-ATL - Alexa546-(AbII-AbI)-HLA-DR). Additional "semi-quantitative" data interpretation would be affected by large approximations, and would also rely on uncertain assumptions. Nonetheless, the measured relative changes of FRET efficiency with time from maturation are intrinsically significant and relevant for the clinical evaluation of immunotherapy vaccination trials.
It has to be pointed out that we chose the acceptor photobleaching FRET method for its complete insensitivity to certain artifacts, including the direct excitation of acceptor. According to this FRET measurement method, both of the images (i.e. the green and in the red channels) were acquired before and after photobleaching through the appropriate emission barrier filters. Moreover, for each sample, three different staining preparations were carried out: +Alexa488 -Alexa546; +Alexa488 +Alexa546; -Alexa488 +Alexa546. All the sample preparations were analyzed before the active photobleaching FRET measurements. Under our experimental conditions, no significant background of Alexa 546 excitation in the absence of Alexa488 was observed. Furthermore, the test presented in Figure 2B (acceptor levels vs. E%) was weighted against the presence of crosstalk artifacts.