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Objective: To establish a method of collecting transplantation major histocompatibility complex (MHC) peptides., Methods: Splenic cells of C57BL/6 donor mice were injected into BALB/c recipient mice. The splenic cells of the recipient mice were soaked in pH3.3 citric acid buffer to wash off the MHC conjugated peptides. Peptide contents and components in the elutriant were detected by BCA protein assay and high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS). Antigenicity of the peptides was verified by murine heterotopic heart transplantation., Results: Fifty micro-gram mixed peptides (including short peptides) per 10(8) cells were collected by the elution method. HPLC-MS/MS revealed that there were differences in peptide components between allo-transplantation group and control group. Medial survival time of heart grafts of same strain MHC peptide pre-immunization group was 8 days, and that of allo-MHC peptide pre-immunization group was 4 days. Medial survival time of heart grafts was shortened significantly by pre-immunization with allo-MHC peptides., Conclusion: Transplantation MHC peptides can be obtained by donor splenic cell immunization and elution from recipients' splenic cells with mild acid. They can be used for screening transplantation antigenic determinants.

Objective: To generate a recombinant Adenovirus encoding a GFP (green fluorescent protein)-report gene and a single-chain trimer of MHC restricted HBsAg CTL epitope., Methods: An oligonucleotide encoding H-2L(d) restricted HBsAg CTL epitope was synthesized and fused with H-2L(d) DNA molecule to construct the eukaryotic expression vector carrying the HBsAg-SCT gene. The HBsAg-SCT gene was subcloned into a GFP adenovirus expression vector,which was transfected into Ad293 cells for packaging and amplification of recombinant adenovirus encoding HBsAg-SCT., Results: HBsAg-SCT has been cloned into an adenovirus vector encoding GFP report gene successfully as confirmed by double enzyme digestion and direct sequencing. HBsAg-SCT was expressed by infected Ad293 cells demonstrated by western blot assay., Conclusion: A recombinant adenovirus expressing HBsAg-SCT and green fluorescent protein report gene has been generated.

Objective: To investigate the influence of glucocorticoid on phenotype of thymic dendritic cells in mice and to investigate the protective effect of Yougui Pill (YGP) on it., Methods: BALB/c mice allocated in the group A and B were treated respectively with 10 mg/kg hydrocortisone, alone and combined with 20.81 g/kg YGP. The control mice were treated with normal saline. The changes before and after treatment of I-A(d) and H-2K(d) antigen presentation molecules expression in CD11c(+) and CD45(+) thymic dendritic cells of mice were analyzed by flow cytometry assay, and the expression of intercellular adhesion molecule-1 (ICAM-1) and leukocyte function-associated antigen-1 (LFA-1) mRNA in thymocytes were determined by RT-PCR as well., Results: The percentage of I-A(d+) and H-2K(d+) in CD11c(+) in Group A after treatment was 46.77 +/- 4.32% and 64.34 +/- 7.69% respectively, as compared with those in the control group (65.81 +/- 7.69% and 31.88 +/- 5.01%), the percentage of I-A(d+) was lower and that of H-2K(d+) was higher significantly (all P <0.01). Meantime, the expression of ICAM-1 and LFA-1 in thymocyte in Group A (30.11 +/- 2.51% and 30.40 +/- 3.77%) was significantly lower than that in the control group (46.35 +/- 3.34% and 47.28 +/- 2.91%) respectively (P <0.01). Changes in Group B showed that treated by hydrocortisone in combination with YGP, the above-mentioned hydrocortisone-induced changes could be obviously reversed, the outcome of CD11c(+) I-A(d+) was 54.19 +/- 5.08%, ICAM-1 33.97 +/- 2.04% and LFA-1 34.80 +/- 2.92%, the difference between the two treated groups in these indexes all showed statistical significance (P <0.05)., Conclusion: Glucocorticoidcan inhibit the expression of major histocompatibility complex class II antigen molecule, but promote the expression of major histocompatibility complex class I in CD11c(+) and CD45(+) dendritic cells, down-regulate ICAM-1 and LFA-1 transcription, while the tonifying yang recipe, YGP, has a dominant protective effect against the above actions of glucocorticoid.

Aim: To study the mechanism of peripheral T cell tolerance induced by superantigen Staphylococus aureus enterotoxin B (SAgSEB)., Methods: Splenocytes from C57BL/6J (B6) mice were stimulated with SEB in-vitro. The proliferation of lymphocytes was determined by MTT colorimetry. The apoptosis of anergic T cells, the cells being in the S and G(0)-G(1) phase and MHC-I (H-2K(b)) expression of T cells were analyzed by flow cytometry (FCM). Apoptosis of anergic T cells was also demonstrated by DNA electrophoresis ladder., Results: SEB could induce strong proliferation of CD4+ T cells and the maximal percentage of CD4+ T cells in S phase appeared on 3rd day after SEB stimulation and then decreased, but the changes of percentage of CD4+ T cells in G(0)-G(1) phase was opposite to that in S phase. CD4+ T cells became anergy on 3rd day and apoptosis of anergic CD4+ T cells appeared on 5th day. Percentage of apoptosis cells increased gradually and could not be reversed by addition of anti-CD3 Ab and ConA. Expression of MHC-I (H-2K(b)) was marginally declined accompanied by CD4+ T cell anergy after SEB exposure., Conclusion: SEB-induced tolerance may have relation to anergy and apoptosis of CD4+ T cells, and down-regulation of MHC-I molecules.

Objective: To predict tumor antigenic peptides from the intact amino acid sequence of tumor antigens, thereby providing a guide to the design and development of tumor peptide-based vaccines., Methods: Antigenic peptides were predicted by using a computer program PEPMOTIF based on MHC class I allele-specific consensus motifs; and a scoring system was established to assess the likelihood of the peptides to bind to MHC class I molecules. MHC class I-peptide binding assay and MHC class I-peptide stabilization assay were used to determine the binding ability and affinity of the predicted peptides., Results: Three peptides derived from mouse mutant p53 were predicted. mp53P132F(LNKLFFQL) was shown to bind to H-2Kb, but not H-2Db. mp53P246S (MGGMNRSPIL) and mp53P270C (GRDSFEVCV) bound to neither H-2Kb nor H-2Db. A relationship was found between peptide scores and binding of the peptides to the relevant MHC class I molecules., Conclusion: The computer program PEPMOTIF combining with a scoring system is a simple and efficient tool for predicting MHC class I binding peptides derived from antigenic proteins.