?(Fig.5).5). profiling of p53-/- thymoma cells, at best results in a poor tumor safety therefore questioning this way of detection of fresh tumor rejection epitopes. Keywords: Tumor connected antigens, mRNA microassay, Peptide, p53, Vaccination Intro Recognition of tumor connected antigens (TAA) identified by CD8+ T cells and the related major histocompatibility complex class I (MHC-I) restricted epitopes has led to peptide-based vaccination methods in experimental animals as well as with clinical settings [1-5]. Since many MHC-I restricted TAA so far recognized represent peptides derived from self proteins, it is not surprising that most of these TAA are relatively weak immunogens and that reports demonstrating tumor regression after peptide vaccination in medical tests are sparse. Occasional marked medical regressions of melanoma have been observed after peptide vaccination [5-7]. To search for new TAA, we have recently used mRNA profiling to analyze a panel of spontaneously arising thymomas in p53-/- mice and recognized a number of upregulated mRNAs [8]. Immunizing having a pool of six peptides representing upregulated RAD50, a part of a DNA regulatory protein complex [9], we acquired partially safety against the take Il1a and growth of inoculated tumor cells overexpressing RAD50 mRNA. This finding suggested to us that tumor rejecting epitopes can be recognized by mRNA manifestation profiling. In the present work we have focussed within the CTL generating effect after immunization with individual RAD50 derived peptides and with H2b-binding peptides derived from additional proteins encoded by differentially upregulated mRNAs [8]. By immunization, half of the peptides, including two of the RAD50-derived peptides, were found to induce significant peptide specific CTL reactions. However, none of these peptides were capable of eliciting CTL reactions against the thymoma cells Tecarfarin sodium from which they were derived. Mice vaccinated with the two immunogenic RAD50 peptides were weakly safeguarded against tumor take, whereas vaccination having a pool of the four immunogenic thymoma connected peptides derived from additional, potentially upregulated thymoma proteins, did not influence tumor take. Treatment having a obstructing antibody against the cytotoxic T lymphocyte antigen CTLA4 [10] offers been shown previously to enhance the Tecarfarin sodium effect of tumor rejection in mice vaccinated with irradiated tumor cells [11,12]. However, this treatment did not increase peptide vaccine-induced safety against tumor take, suggesting the tumor connected peptides, characterized in the present study, represent at best very poor tumor rejection epitopes. Results Generation of CTL reactions Individual RAD50 derived peptides [8] (observe Table ?Table1),1), having a binding affinity (KD) for H-2b at 12C280 nM [13], were injected subcutaneously in Freunds Incomplete Adjuvant (FIA). Splenocytes were recovered 10 days after immunization and challenged in vitro for 5 days with irradiated syngenic spleen cells pulsed with specific peptide (observe Materials and Methods). CTL reactions were measured against RMA-.S and SM7 target cells pulsed with specific peptide or mock peptide. Mean data for groups of three immunized mice are demonstrated in Figure ?Number1.1. Only two of the RAD50 peptides, RAD23C31 and RAD24C31, with KD ideals of 280 and 70 NM respectively [13], induced a CTL response (Fig. ?(Fig.1A)1A) and only immunization with the RAD23C31 peptide induced killing of RAD24C31-pulsed SM7 cells (Fig. ?(Fig.1B),1B), whereas unpulsed SM7 cells were not killed by any of the peptide specific CTLs (Fig. ?(Fig.1C).1C). Experiments (not included) showed that CTLs raised against the RAD23C31 peptide killed RAD24C31 pulsed RMA-S cells to the same lengthen as RAD23C31 pulsed cells whereas CTLs generated against the RAD24C31 peptide killed the RAD23C31 pulsed RMA-S with only half the effectiveness of RAD24C31 peptide pulsed cells. These data suggest that some of the CTLs generated against the RAD23C31 peptide separately identify the C-terminal glutamine in the RAD23C31 peptide. Open in a separate window Number 1 CTL development in vivo against SM7-derived RAD50 peptides. Groups of three mice were immunized once subcutaneously with individual peptides including a helper peptide. Splenocytes were peptide challenged ex lover vivo at day time 10 and assayed for CTL activity five days later. A, Generation of CTLs against RAD50 peptides. B, CTL reactivity against SM7 thymoma cells pulsed with the RAD50 peptides. C, CTL reactivity against non-pulsed SM7 thyoma cells (observe Table ?Table11 for peptide name, sequence and MHC-I binding affinity). Table 1 Potentially overexpressed p53-/- SM7 thymoma proteins as analyzed by mRNA manifestation profiling including expected, sequenced and assayed protein-derived H2b binding peptidesa.
Protein namePeptide sequencePeptide nameKD nMbRAD50RQIKNFHELRAD50 391C39945RAD50SQQRNFQLLRAD50 603C61112RAD50SAEQNKNHIRAD50 1257C126513RAD50IISFFSPLRAD50 24C3170RAD50QIISFFSPLRA50 23C31280RAD50AIMKFHSMRAD50 1131C113936Endonuclease*YAYTFWTYMEncl261C26980RAD23KALGFPESLRAD23 328C3362950PMS2LGQFNLGFIPMS2 676C684410PMS2*FGPQDIDELPMS2 775C7832Cathepsin B*FYNVDIDYLCatB 47C55155TranslinVSEIFVELTranslin 3C10560Protease-nexin 1*WHEPFFILPn1 3C10NDProtease-nexin 1VHSQFNSLPn1 18C259500 Open in a separate window afor Tecarfarin sodium details observe research no. [8]bdata are from ref. [13]. *Immunogenic peptides, observe Figs ?Figs11,?,22. In independent.