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Javorovic, Miran (2004): T-Cell Stimulation by Melanoma RNA-Pulsed Dendritic Cells. Dissertation, LMU München: Faculty of Biology
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Abstract

In situations where well-established approaches such as surgery, radiation therapy and chemotherapy fail to help cancer patients, immunotherapy has the potential to be an effective alternative. Tumour cells can sometimes be distinguished from corresponding normal cells due to their expression of tumour-associated antigens (TAAs), most of which are unaltered self-molecules. These molecules must be presented to the immune system in the context of danger in order to achieve their specific recognition. If dendritic cells (DCs), the most potent professional antigen-presenting cells, are loaded with RNA, they will translate the RNA into protein, process the protein into peptides and present the peptides within MHC molecules (pMHC) on their surface to cytotoxic T-lymphocytes (CTLs) and T-helper cells in a stimulatory manner. These effector cells can, in turn, recognise tumour cells. The goal of these studies was to find optimal conditions for producing a DC-based vaccine for cancer patients using TAAs in the form of RNA. The studies were designed to quantitate RNA transfer into DCs, to determine the intracellular stability of transfected RNA in DCs and to analyse the kinetics of protein expression and the generation of functional pMHC ligands that could activate effector memory CTLs. Simultaneous activation of CTLs with specificities for different antigens minimises the potential for tumour escape through immune selection of tumour variants showing loss of individual antigens. Thus, generation of multiplex pMHC ligands for CTLs may improve clinical efficiency. On the other hand, peptide competition for MHC molecules within the DC may limit pMHC ligand generation. This central immunological question was addressed by comparing DCs loaded with total cellular tumour RNA, amplified total cellular tumour mRNA and pools of defined single-species tumour-antigen cRNAs versus individual single-species tumour-antigen cRNAs for their capacity to display various pMHC ligands and activate CTLs of corresponding specificities. Experiments performed with RNA encoding the enhanced green fluorescence protein (EGFP), a reporter protein, showed that the highest efficiency of RNA transfection into DCs was achieved with electroporation, reaching levels of 90% positive cells. The fact that mature DCs expressed more EGFP than immature DCs suggests that this stage of DC maturation will be optimal for vaccine development. Importantly, electroporation and RNA transfer did not alter the expression of antigen-presenting and co-stimulatory molecules on the surface of DCs. The melanoma model was chosen for extensive analyses because its characterisation at the cellular and molecular levels has made it a very informative model for understanding cancer immunity. In addition to total cellular melanoma RNA, single-species cRNAs were used encoding the melanoma-associated antigens, tyrosinase, Melan-A and CDK4-R24C. Antigen presentation was detected with the help of effector memory CTL clones specific for each of these antigens. The CTL stimulatory capacity of RNA-transfected DCs was higher if they were allowed one day to recuperate from electroporation and to produce pMHC complexes. Tyrosinase cRNA dose-finding showed that more RNA would indeed result in higher stimulatory capacities of transfected DCs. Kinetics of tyrosinase cRNA degradation, similar to kinetics of EGFP cRNA degradation, revealed that the amount of transfected RNA rapidly decreased inside the DCs within 1.5 hr after electroporation. The smallest decrease was observed with the highest amount of RNA applied in electroporation. The kinetics of RNA degradation and protein half-life will be important parameters to consider in defining the right time-frame for T-cell activation by engineered DCs. When reverse transcription PCR (RT-PCR) was performed with total cellular melanoma RNA samples to generate amplified mRNA, the Melan-A, tyrosinase and CDK4/CDK4-R24C message was amplified 62-fold, 24-fold and 2-fold, respectively. These differences likely reflect variations in the expression levels of the corresponding antigen message in melanoma cells from which the RNA was isolated. Approximately 17240-fold more CDK4-R24C message, at least 500-fold more tyrosinase message and at least 480-fold more Melan-A message was found in single-species cRNAs when the same masses of single-species cRNA and amplified melanoma mRNA samples were compared. This explained why electroporation of single-species cRNAs into DCs yielded the highest DC stimulatory capacities. Combinations of tyrosinase, Melan-A and CDK4-R24C cRNAs were studied for their capacity to induce satisfactory levels of T-cell stimulation when presented by DCs. Here it was demonstrated that antigen competition was not a critical factor, since CTL responses to pooled RNAs were not inhibited even though competition for MHC class I molecules may have occurred within the DCs. DCs also developed CTL stimulatory capacities, but at much lower levels, using amplified melanoma mRNA. Two antigen-specific CTL clones displayed higher reactivities upon exposure to pMHC produced naturally by RNA-transfected DCs than to synthetic peptides pulsed onto DCs. In one case, this could be explained by a post-translational modification of the peptide, which normally occurs within cells. Since this particular modification was not represented in the synthetic peptide, which was chosen from the protein sequence, the synthetic peptide was not well recognised. This demonstrated that the use of RNA technology eliminates the need to know the correct sequences of immunogenic peptides. Thereby, DCs are better than scientists at choosing antigens and their epitopes for presentation to T-cells. These data provided a better understanding of antigen presentation by DCs based on the use of RNA, giving insight into antigen competition and paving the way for the use of pooled RNAs of defined species for the development of a multiplex vaccine. They also allowed a precise protocol for efficient T-cell activation to be defined. Further experiments will demonstrate whether quantitative differences detected in antigen presentation between DCs loaded with total cellular tumour RNA and amplified total cellular tumour mRNA versus single-species tumour-antigen cRNAs have an impact on de novo T-cell priming in vitro and in vivo.