Whether RNA should be transfected into immature or mature DCs depends on many factors. DCs of different maturation stages may display distinct susceptibilities to RNA transfer. Furthermore, immature and mature DCs are responsible for different steps of antigen processing and presentation, meaning that antigen in the form of RNA must be introduced into cells with proper functionality. It is clear that mature DCs most efficiently present antigenic peptides due to their up-regulation of MHC expression, improved rate of peptide loading into MHC molecules and intensified transport of pMHC complexes to the cell surface. However, at exactly which stage before antigen presentation DCs generate the epitope peptides is the matter of considerable controversy.
Peptides are largely produced when cytosolic proteins are degraded by proteasomes. In DCs, changing profiles of MHC-bound peptides are associated with the replacement of active proteasome subunits by the so-called immunosubunits (Gileadi U et al. 1999). Both
proteasomes and immunoproteasomes seem to have similar efficiencies in degrading proteins, but bioinformatic analysis of experimental degradation data showed that they had
individual cleavage motifs, yielding a different and less diverse pool of peptides produced by immunoproteasomes (Kesmir C et al. 2003). A less diverse pool of peptides should
enhance the presentation of relevant antigenic peptides, since they do not have to compete for MHC molecules with many other irrelevant peptides. If proteasomes were restricted to immature DCs and immunoproteasomes to mature DCs, the variable quality of generated peptides might even serve to prevent autoimmune reactions. However, data regarding the time point of immunoproteasome assembly and activation are somewhat contradictory and do not yet allow clear conclusions to be drawn. One group reported down-regulation of mRNA encoding the immunoproteasome subunits upon DC maturation (Li J et al. 2001).
This might contribute to the higher antigen-processing capability of immature DCs. Another group found that immunoproteasomes already represented half of the proteasome population in immature DCs. Quantitative analysis of mRNA showed either very limited or moderate induction of immunoproteasome subunit expression in mature DCs. Given that proteasomes are long-lived complexes with half-lives of several days, it was postulated that their replacement with immunoproteasomes occurs only gradually over an extended period of time. Therefore, the amount of immunoproteasomes may not be significantly different between immature and mature DCs and should not strongly influence antigen processing. However, authors of the study postulated that other, unidentified mechanisms involving regulation of (immuno)proteasome activity are responsible for the marked differences in antigen presentation between immature and mature DCs (Macagno A et al. 2001). The DC maturation stage at which such regulatory
mechanisms are active remains unclear.
Obviously, the need is to introduce RNA into DCs at a maturation stage that allows the newly synthesised proteins to be degraded by immunoproteasomes and not by proteasomes. Until more precise information is available about when immunoproteasomes are activated inside DCs, the decision of whether to transfect RNA into immature or mature DCs can only be based on transfection and antigen-presenting efficiencies. Unfortunately, relevant data are scarce and also contradictory. Van Tendeloo and colleagues obtained the most potent CTL activation when immature DCs were loaded with Melan-A cRNA by electroporation or lipofection, subsequently matured with LPS and TNF-α, and then co-incubated with the CTLs. They attributed the lower stimulatory capacity of DCs transfected after maturation to a lower degree of transfectability by either lipofection or electroporation (van Tendeloo VF et al. 2001). My results contribute to the
controversy by showing that electroporation of EGFP cRNA was more efficient with mature DCs, compared to immature DCs (Figure 4.2B). Furthermore, mature DCs expressed more protein than immature DCs, based on the use of identical amounts of electroporated or lipofected EGFP cRNA (Figure 4.3). This was not a function of transfection efficiency because mature DCs were more difficult to lipofect (Figure 4.2B), but nevertheless expressed more EGFP than their immature counterparts. If mature DCs translate RNA into more protein, they are likely to generate more peptides. Therefore, in my experimental system tumour-antigen RNA was transfected into mature DCs only, resulting in satisfactory levels of CTL stimulation.
As will be discussed below, protein expression decreases relatively quickly after transfection due to degradation of transfected RNA by intracellular RNases, thereby narrowing the time period in which antigens are produced and processed. Different susceptibilities of proteins to digestion by intracellular proteases also have to be taken into account. The half-life of tyrosinase was estimated to be at least 12 hr (Halaban R et al.
1997) and that of Melan-A to be approximately 3 hr (de Mazière AM et al. 2002). The
relative instability of Melan-A was indicated in this study when Melan-A cRNA- transfected DCs were stained with a Melan-A-specific antibody and analysed in FACS™
(Figure 4.25). Melan-A was expressed in 88% of the cells from one donor 7 hr after electroporation. However, 24 hr after electroporation, Melan-A was detected in 66% of transfected DCs from a second donor. Additional staining experiments, to assess precise kinetics and quantify mean fluorescence intensity, are required to confirm the observed decrease in Melan-A expression. In terms of antigen presentation, different stabilities of TAAs could lead to a situation in which peptides originating from one antigen, e.g. Melan- A, are generated and presented sooner than peptides derived from another antigen, e.g. tyrosinase. Considering the fact that an average pMHC class I complex has a half-life of 5-10 hr on the cell surface (Cella M et al. 1999), choosing the right time-frame for DC-
CTL interaction may be critical for efficient T-cell activation. Whereas selection of an optimal time point may not be possible for all antigens encoded by total cellular RNA, it might prove to be advantageous with single-species tumour-antigen cRNAs. Therefore, an additional argument in favour of electroporating mature DCs might be that the expression of their co-stimulatory, adhesion and antigen-presenting molecules is already up-regulated. This phenotype is not changed by electroporation (Figure 4.5). If mature DCs are transfected, there is no need to wait an additional 1-2 days in order for their maturation to
take place. Theoretically, they can be used for CTL stimulation immediately after transfection. Practically, however, DCs need time to recuperate from the shock of electroporation and to produce, process and present the antigen. This was demonstrated by my finding that tyrosinase cRNA-pulsed DCs developed a higher stimulatory capacity when they were cultured one day before contact with the CTLs, as opposed to only two hours (Figure 4.13). This 24 hr time period might be used for simultaneous maturation, if immature DCs were electroporated, however, if RNA encoding a short-lived antigen is used, it may be necessary to initiate co-incubation of DCs and CTLs sooner, thereby limiting the time available for DC maturation.
Keeping in mind that all steps performed in vitro are merely attempts to simulate in vivo
situations, a strong argument in favour of RNA transfection into immature DCs is made by the Gilboa group (Nair S et al. 2003). Instead of maturing DCs in vitro through culture
with various agents that mimic the conditions encountered at a site of inflammation, immature DCs were electroporated and then injected into adjuvant-pretreated skin. Thereby, transfected DCs were matured in situ and displayed migratory capacities,
immunostimulatory properties and anti-tumour activity that were comparable to, or even better than, those achieved using DCs matured ex vivo. Mature DCs injected into adjuvant-
treated sites migrated more efficiently, but their immunostimulatory capacities remained lower than those of immature DCs that matured in situ. In the end it will probably be
necessary to evaluate these various parameters using labeled DCs in comparative clinical trials in order to define the best clinical strategy.