Interestingly, R788 the avidity of response to the OVA was similar (1.7×10−9 M) to the response to TRP2 (1.3×10−9 M) suggesting that there is no deletion of the

repertoire to this self Ag. However responses to both epitopes could be increased over 100-fold, by using an Ab–DNA vaccine compared to peptide immunization. These results suggest that at the each peptide MHC complex interacts with a defined number of TCR within the repertoire playing an important role in determining the original avidity 28 but this can then be further modulated at the clonal level. The range of avidities observed in the mice analyzed spans five logs, yet within individual experiments this variation is less. This probably reflects the plasticity of the avidity to any given TCR:MHC/peptide combination with optimal immunization leading to a high avidity. The avidity with DNA vaccination depends upon the degree of direct v cross presentation, GSK-3 beta pathway which may vary between experiments. However this does not explain the reduced variability within one

experiment. Our explanation is that despite careful operating procedures, this is related to the efficacy of immunization/monitoring of the response. We are aware that timing for harvesting the splenocytes to plating into an assay is a key parameter and endeavor to keep this constant. Finally experiments were performed over a 2-year period and factors such as subtle changes in mice, environment and batches of DNA have to be considered. Within the small groups these factors would be more consistent. The avidity of the responses to the TRP2/HepB human IgG1 DNA vaccine varied from 5×10−13 M to 5×10−8 M in different mice but was on average

5×10−10 M. Is this avidity sufficient to result in effective immune response? An elegant study by Dutoit et al. demonstrated that T cells cloned from cancer patients exhibited an exponential increase in killing with T-cell avidity greater than 10−9 M Ureohydrolase 2. A similar study with T-cell clones showed that only high-avidity clones adoptively transferred caused tumor rejection in mice 1. The avidity resulting in tumor killing will depend upon the expression level of the Ag/MHC. Our study is in agreement with these demonstrating that selective vaccination can increase avidity to a level sufficient for therapy. The frequency and avidity of the responses from human IgG1 DNA immunization was significantly higher than that observed from peptide immunization. Initially unlinked peptides were used but due to lack of T-cell help, these gave very weak responses (results not shown). To give a more reasonable comparison, the CTL epitopes were linked to a well known helper epitope which still gave poor responses. This was perhaps not surprising as even linked helper-CTL peptides have a very short half life and are poor immunogens in vivo29.