| Lymphoid malignancies, which belong in hematological malignancies, can be devided into two categories:Hodgkin’s lymphoma (HL) and Non-hodgkin lymphoma (NHL). Chemotherapy combined with radiotherapy can effectively suppress HL, but shows poor effect on NHL. Therefore, immunotherapy is attracting increasing attention in the academic research area.T cells can be divided into two subgroups based on their expression of T cell receptor (TCR) on the cell surface, i.e., the a(3T lymphocytes andγδT lymphocytes.γδT cells can directly recognize stress-inducible proteins and tumor-associated antigens without MHC restriction, and show significant cytotoxicity against autologous, allogeneic and xenogeneic tumor cells. They are increasingly recognized as the candidate cells for adoptive immunotherapy, and become the focus in the area of tumor immunotherapy. Because of the low frequency (<10%) of y8T cells in CD3 T cells in the peripheral blood, to obtain sufficient quantities ofγδT cells with significant anti-tumor effect becomes a bottleneck forγδT cell-based adoptive immunotherapy.In the present study, we expandedγδT cells with immobilized anti-TCRγδantibody and obtained great quantities of yST cells with high purity and relative intact TCR spectrum. In order to adapt anti-TCRγδantibody expandedγδT cells for clinical application, we carried out preclinical safety and efficacy evaluation, which provided experimental basis for the treatment of lymphoid malignancies using autologousγδT cell-based adoptive immunotherapy.First, we established the quality and safety monitoring system forγδT cells expanded with immobilized anti-TCRy5 antibody, observed the survival rate, stability and bioactivity ofγδT-cell preparations, and detected bacterium, endotoxin and residual mouse IgG in the finalγδT-cell preparations. In the present study, all the testedγδT-cell preparations met the pharmacopoeia requirements for biological products with-90% survival rate,～80%purity, satisfactory stability, and significant cytotoxicity against both solid and hematological tumor cells.γδT-cell preparations were sterile and endotoxin-free, with residual mouse IgG less than lng/ml.Second, we optimized the culture conditions forγδT cells. The metabolism of IL-2, which is necessary for the culture ofγδT cells, was monitored to determine the time interval for IL-2 supplementation in the culture process. Different media (with or without autologous serum) were used to cultureγδT cells. Survival rate, purity, proliferation and biological function ofγδT cells were examined and compared. The medium most suitable for clinical applications was chosen.Last, the effectiveness ofγδT cells expanded with immobilized anti-TCRγδantibody in treatment of lymphoma was evaluated. The most commonly used method forγδT cells expansion use phosphoantigen as stimulus. Compared withγδT cells expanded with phosphoantigen,γδT cells expanded by immobilized anti-TCRγδantibody expressed similar bioactive molecules, even showed comparable cytotoxicity against lymphoma cells. However, different fromγδT cells expanded with phosphoantigen, antibody expandedγδT cells contained V81 subset. In the present study, Vδ1 T cells showed similar cytotoxicity against lymphoma cells compared with Vδ2 T cells. Notably, the expanded Vδ1 T cells manifested an enhanced migration tendency towards CCL17 and CCL22, the two chemokines which are expressed by various lymphoma cells. This result indicates a non-redundant role of the antibody expandedγδT cells in anti-lymphoma immunotherapy. In addition, the antibody expandedγδT cells showed potent cytotoxicity against all of the tested lymphoma cells in vitro. Treatment of Daudi-lymphoma-bearing nude mice further confirmed their significant anti-lymphoma effect in vivo. Peripheral bloodγδT cells from 12 lymphoma patients efficiently proliferated in response to immobilized anti-γδTCR antibody, which further proved the feasibility ofγδT cells in the treatment of lymphoma. Severer acute respiratory syndrome (SARS) is caused by a novel coronavirus. SARS associated coronavirus (SARS-CoV), is a single chain RNA virus with structural proteins including the spike glycoprotein (S), membrane protein (M), envelope protein (E) and nucleocapsid protein (N). The major immunogens include spike glycoprotein and nucleocapsid protein, which are the research focuses of this study.We had previously identified five linear B-cell epitopes from a 10-mer overlapped peptide library, which were highly cross-reactive with all tested SARS convalescent patients’sera. Five linear B-cell epitopes had been randomly assembled into strings joined by (G4S)3 linkers. Gene fragments encoding the designed strings had been obtained by overlap extension polymerase chain reaction or de novo DNA synthesis, and cloned into prokaryotic expression vectors. In the present study, we expressed and purified two of the recombinant poly-epitope peptides (rPEP1 and rPEP2). Then the binding specificity of the rabbit anti-rPEP1 and anti-rPEP2 polyclonal antibodies was evaluated by hybridizing with rPEP1 and rPEP2 in Western blotting. Subsequently, the SARS-CoV-neutralizing activities of the rabbit anti-rPEP1 and anti-rPEP2 polyclonal antibodies were evaluated by pseudovirus neutralization assay. Our results suggest that recombinant poly-epitope peptides rPEP1 and rPEP2 possess high immunogenicity and virus-neutralizing ability, which will provide experimental basis for the preparation of poly-epitope peptide vaccine. |
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