PMC:3724992 / 11713-18507 JSONTXT

{"target":"https://pubannotation.org/docs/sourcedb/PMC/sourceid/3724992","sourcedb":"PMC","sourceid":"3724992","source_url":"https://www.ncbi.nlm.nih.gov/pmc/3724992","text":"Results\n\nPrediction of putative CTL epitopes restricted with HLA-A*0201\nTo predict the HLA-A*0201-restricted CTL epitopes of neuritin, two programs (BIMAS and SYFPEITHI), were used to scan the complete amino acid sequence of this antigen. Four highest-scored 9-amino-acid peptides were chosen as candidates for further identification (Table 1). These peptides were chemically synthesized, purified, and identified. The molecular weight of each peptide determined by mass spectrometry assay was similar to its theoretical molecular weight, and the purities of these peptides were all \u003e95 % (data not shown).\nTable 1 Predicted neuritin epitopes binding to HLA-A2.1\nPosition Length Sequence BIMAS score SYFPEITHI score\n13–21 9 ILAVQIAYL 459.398 30\n121–129 9 LLPAFPVLL 138.001 25\n4–12 9 KLNGRYISL 112.153 26\n127–135 9 VLLVSLSAA 71.872 25\n\nMHC peptide-binding and stability assay\nThe binding affinity and stability of these peptides to HLA-A2.1 was determined by using antigen processing-deficient T2 cells because their enhanced HLA-A2.1. As shown in Table 2, all of the peptides synthesized were bound to HLA-A2.1 molecules but with different affinity and stability. Of four peptides selected, neuritin13–21 up-regulated the HLA-A2.1 molecular expression and showed high affinity and stability to HLA-A2.1, whereas neuritin121–129 and neuritin4–12 showed moderate affinity and neuritin127–135 only had low affinity and stability to the molecule.\nTable 2 HLA-A2-binding affinity and stability of neuritin-derived peptides\nPosition Length Sequence FI DC50\n13–21 9 ILAVQIAYL 1.68 \u003e8\n121–129 9 LLPAFPVLL 1.24 6–8\n4–12 9 KLNGRYISL 1.07 6–8\n127–135 9 VLLVSLSAA 0.68 2–4\n519–526 9 FSYGFFV 0.38 \u003c2\nThe shaded region is the control peptide values\n\nExpression of neuritin in target cells\nThe expression of neuritin mRNA and protein in cell lines in this study was analyzed by RT-PCR and Western blot. As shown in Fig. 1, neuritin mRNA and protein were detected in U251 and U87 cell lines. However, neuritin mRNA and protein could not be detected in MCF-7 and autologous lymphocytes.\nFig. 1 Expression of neuritin in different target cells. Total RNA was isolated from tumor cell lines using Tripure Isolation Regent Kit. Two microliters RT product was amplified with PCR by using TaqDNA polymerase (using standard procedures). RT-PCR products were then run on a gel and visualized with ethidium bromide. For Western blot analysis, proteins in the cell extracts were separated by SDS-PAGE and were then analyzed with anti-neuritin MAb (antibodies-online company). 1 U251 cells; 2 U87 cells; 3 MCF-7 cells; 4 autologous lymphocytes\n\nEnzyme-linked immunospot (ELISPOT) assay for IFN-γ\nSince CTLs are known to produce the Th1 cytokine IFN-γ, peptide-specific T cells were enumerated by measuring IFN-γ-producing cells by ELISPOT assay. As shown in Fig. 2, neuritin13–21, neuritin121–129 and neuritin4–12 peptides were found to generate a strong peptide-specific T cell response by virtue of their ability to induce increased frequencies of IFN-γ-producing T cells, as compared to the negative peptide control. These results suggest that neuritin peptide vaccines can increase IFN-γ secretion by effectors and enhance the Th1 immune response.\nFig. 2 Specific IFN-γ by ELISPOT assay. The PBMCs of human HLA-A2+ donors were obtained and then cultured in RPMI 1640 supplemented with 10 % FCS, 100 U/ml penicillin, and 100 μg/ml streptomycin. Dendritic cell were generated, and loaded with different peptides at a final concentration of 100 μg/ml for 4 h and were then irradiated with 20 Gy, which prevented all outgrowths in the control cultures. Autologous T cells were restimulated every 7 days with the peptide-pulsed DCs to generate peptide-specific CTLs. The IFN-γ secretion was then assessed on day 23. Experiments performed in triplicate showed consistent results. Compared with controls, P \u003c 0.05\n\nInduction of CTLs efficiently in vitro\nPBMCs from four HLA-A2.1+ donors were stimulated with synthetic peptides with the previously published method for CTL induction [19]. Of four tested, neuritin13–21, neuritin121–129 and neuritin4–12 peptides were able to elicit neuritin-specific CTLs, which could lyse target cells expressing neuritin and HLA-A2.1 (Fig. 3).\nFig. 3 Specific lysis of CTLs against target cells. Target cells were incubated with 51Cr (100 μCi per 1 × 106 cells) for 2 h in a 37 °C water bath. After incubation with 51Cr, target cells were washed three times with PBS, resuspended in RPMI-1640 medium, and mixed with effector cells at a 25:1, 50:1 or 100:1 of effector to target (E/T) ratio. After a 4-h incubation, the supernatants were harvested, and the amount of released 51Cr was measured with a gamma counter. Compared with controls, P \u003c 0.05\n\nInhibition of the recognition of effectors by anti-HLA2 antibody\nTo determine whether the peptides induced effectors recognized target cells in an HLA-A2-restricted manner, the mAbs against HLA-A2 were used to block recognition by effectors. Our results showed that the anti-HLA-A2 antibody could significantly eliminate the cytotoxicity of the effectors against neuritin and HLA-A2 positive cells (Fig. 4), which implied that the induced effectors lysed target cells in an HLA-A2-restricted manner.\nFig. 4 Inhibited recognition of induced cells by anti-HLA-A2 antibody. Target cells were incubated with 100 μl anti-HLA-A2 antibody (functionally blocking mAb, from BB7.2 cell hybridoma supernate) for 1 h at 4 °C. The cytotoxic activities of CTLs were determined against target cells at various E/T ratios using 51Cr release assay. Experiments performed in triplicate showed consistent results. Compared with controls, P \u003c 0.05\n\nIn vivo induction of epitope-specific CTLs in vivo\nWe investigated whether peptides could induce immunity in vivo. HLA-A*0201/Kb mice were immunized with 100 μg of various peptides prepared in incomplete Freund’s adjuvant (IFA). The cytolytic assay showed that CTLs primed from neuritin13–21, neuritin121–129 and neuritin4–12 immunized mice could lyse neuritin and HLA-A2.1 positive cells with high efficiency (Fig. 5). These results suggested that the peptides could also achieve higher immunogenicity in vivo.\nFig. 5 In vivo induction of epitope-specific CTLs in vivo. HLA-A*0201/Kb mice were immunized with 100 μg of various peptides prepared in incomplete Freund’s adjuvant (IFA) and boosted once a week for three times. As a control, mice were injected with an IFA emulsion without peptide. 7 days after immunization, splenocytes from injected animals were cultured and used as effector cells. The cytotoxic activities of CTLs were determined against target cells at various E/T ratios using 51Cr release assay. Experiments performed in triplicate showed consistent results. 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