Inducing And Screening Of PR1-Specific Cytotoxic T Lymphocytes In Vitro

Chronic myeloid leukemia is one of myeloid malignancies. And there is still no effective treatment at present.The root cause of its relapse is leukemia minimal residual disease (MRD).T cell adoptive immunotherapy may be a promising strategy for providing valuable T lymphocytes in the treatment of tumors and other immune-mediated diseases.However, the specificity, affinity and amount of T cells hampers its applications. How to obtain specific, efficient, a certain number of T cells is the question to be settled urgently. Therefore, strategies were developed to genetically transfer tumor specific immune receptors into patients T cells. The specific and high affinity T cell receptor will be transferred into target T cells by gene transfer.And the transferred T cells will specific lysis tumor cell and product cytokine.Proteinase 3 is one of the human neutrophil granule formation associated differentiation antigens.The original poorly differentiated myeloid leukemia cells in myeloid leukemia patients persistently express high levels of proteinase 3, suggesting that proteinase 3 is one of myeloid leukemia associated antigens.PR1, an HLA-A2 restricted peptide, sequence VLQELNVTV, is from protease 3 . PR1 restrictive CTLs can lyse myeloid leukemia cells, and its lysis capability is significantly related to the expression level of proteinase 3. But it doesn't damage normal bone marrow cells. The purpose of our study is to preparate soluble HLA-A*0201/PR1 tetramer, optimize the conditions of obtaining high-affinity T cells by PR1 peptide stimulate, and screen high-affinity PR1-specific T cells by tetramer technology and flow cytometry.This will be beneficial to chronic myeloid leukemia adoptive therapy and clearance of leukemia minimal residual disease.1 Preparation of soluble HLA-A * 0201/PR1 tetramerWe have been successfully constructed heavy chain pQE3.1-HLA-A2-BSP and light chain pQE3.1-β2m at the preliminary work in our lab.Both of them can be substantially expressed in the M15 strain after induced. The recombinant HLA-A*0201-BSP (BirA substrate peptide) fusion protein as heavy chain andβ2-microglobu1in (β2 m) as light chain were expressed highly as insoluble aggregates in Escherichia coli and then purified with washing inclusion bodies.The two subunits were refolded to form an HLA-A*0201-peptide complex by dilution method in the presence of an antigenic peptide PR1, a HLA-A2-restricted peptide from proteinase 3. The formation of HLA-A*0201 /peptide complex monomers after restoration were identified by indirect ELISA assay with HLA-I conformation-specific W6/32 monoclonal antibody. Then, the monomers were purified by Sephacryl-S300 column chromatography.Refolded HLA-A * 0201/PR1 complex was biotinylated using a BirA enzyme and purified by Sephacryl-S300 column chromatography as well. Biotinylated HLA-A*0201/peptide complex monomer was labeled with streptavidin (including four biotin sites)fluorescein (phycoerythrin or allophycocyanine) by the ratio of 4:1 to form soluble HLA -A * 0201/PR1 tetramer. This experiment succeeded in soluble HLA-A * 0201/PR1 tetramer for markers of antigen-specific T cells.It provided an important instrument for screening CML-related PR1-specific T cells.2 Inducing of PR1- Specific Cytotoxic T Lymphocytes in vitroFirst, we prepared T2 cells loaded with PR1 peptide as stimulating cells. Then we detected peripheral blood lymphocytes with soluble HLA-A*0201/PR1 tetramer, which were stimulated by T2 stimulating cells.It confirmed that the tetramer was against PR1 peptide epitope and could be used to analyze or select PR1-specific T lymphocytes.Second,we stimulated the HLA-A*0201-positive human peripheral blood lymphocytes by use of mixed lymphocyte culture and optimized the stimulation time, the concentration of antigen to stimulate the cells and the proportion of stimulating cells and response cells. It found that we could obtain more PR1-specific CTLs when the concentration of PR1 peptide was 0.5μmol/L or 5μmol/L, the stimulating time was 21 days, and the ratio of stimulating cells and response cells was 1:10. The function of PR1-induced specific cytotoxic T cells was identified by LDH release test .It showed that the ability of PBL induced by PR1 peptide in vitro lysing target cells markedly enhanced.It confirmed that the PR1-specific cytotoxic T cells own the capacity of specific lysing target cells. This is foundation to our follow-up work and chronic myeloid leukemia adoptive immunotherapy.