Tumor Rejection Effects Of Allo-restricted CD4~+T Cells Induced By Tumor-peptide/HLA-DR15 Dimer On Human Cervical Cancer Cell Xenografts In Nude Mice

T cell plays a major role in allogeneic graft rejection and graft-versus-host disease (GVHD), both CD4+ and CD8+ T cells are involved in the process. The ligands recognized by T cell receptor (TCR) are formed by antigenic peptide associated with MHC class I or classⅡmolecule, which are recognized by CD8+ T and CD4+ T cells, respectively. Direct T-cell allo-recognition is the principal player in the initial vigorous immune response to allogeneic cells that causes acute rejection. Current studies indicate that allogeneic T-cell responses, like syngeneic T-cell responses, are largely peptide-specific, and that TCRs interact with allogeneic MHC in a manner that is almost indistinguishable from conventional recognition of antigenic peptide presented by self-MHC. The allo-reactive T cells of peptide-dependent and allogeneic MHC-restricted properties are referred to as allo-restricted T cells. It has been observed that CD4+ but not CD8+ T cells are essential for allo-rejection, since CD4+ T cells are generally regarded as helper cells by facilitating other lymphoid cells involved in immunity, as well as effector cells of graft rejection. Meanwhile, it is well-established that leukemia patients can benefit from a graft-versus-leukemia reaction (GVLR), and evidence of graft-versus-tumor reaction (GVTR) has also been reported in many solid tumors, in which donor T lymphocytes mount an immune response against recipients' leukemia or tumor cells. Both CD4+ T cell and CD8+ T cell are required for GVLR, although most studies are focused on the role of CD8+ T cell in GVLR or GVTR and reveal CD8+ T cell is an effective means. Based on the importance of CD4+ T cells in both nominal and allogeneic responses, we suppose that allo-reactive CD4+ T cells, especially allo-restricted CD4+ T cells, would be responsible for GVLR or GVTR. The aim of this study is to set up a strategy to generate allo-restricted CD4+ T cells and observe tumor rejection effects of the CD4+ T cells.The tumor-peptide/HLA-DR15/IgGl-Fc dimer was prepared in this study, then the tumor-peptide specific allogeneic CD4+ T cells were induced by the dimer-loaded monocytes co-culturing with autologous HLA-DR15 negative PBLs. The tumor-peptide specific allogeneic CD4+ T cells adoptively transferred into nude mice challenged by human cervical cancer cells can inhibit the growth of the tumor.1. Generation of HLA-DR15/IgGl-Fc molecule and its binding to HLA-DR15 negative (DR15-ve) monocytesThe cDNA coding for the extracellular domains of DRa and DRβas well as the leucine zipper motifs of Fos and Jun were cloned by RT-PCR from SiHa cell which is HLA-DR15 genotype. The extracellular domains of DRa and DRβwere joined with the leucine zipper motifs of Fos and Junm, then the Fc segment of human IgGl was attached to DRa-Fos through the EcoR I restriction site. The recombinant plasmid named pFastBacTM Dual+[DR15/IgGl-Fc] was constructed and transformed to Escherichia coli DH10BacTM and the recombinant shuttle plamid Bacmid+[DR15/IgG1-Fc] was obtained. The shuttle plamid was transfected into the insect cell line Sf9 by lipofection and the expression of HLA-DR15/IgG1-Fc dimer (DR15 dimer) was detected by sandwich ELISA and Western-blotting. The results of ELISA and Western-blotting showed that DR15 dimer molecule consists of two parts:the dimerization of extracelluar fragment of HLA-DR15 molecule which can bind to specific T cell receptor (TCR) and the Fc fragment of IgG1 which binds to FcyRI on the cell membrane of monocytes with high affinity.DR15-ve monocytes incubated with purified DR15 dimer and the capacity of DR15 dimer binding to monocytes was confirmed by flow cytometry (FCM). The result showed that the percent of DR15 phenotype monocytes which were DR15-ve genotype significantly increased after incubated with purified DR15 dimer, which indicated that DR15 dimer has the capacity of binding to DR15-ve monocytes.2. Raising peptide-specific allogeneic CD4+ T cells from DR15-ve PBLs co-cultured with autologous monocytes loaded with the dimer DR15-ve PBLs were co-cultured with the dimer-loaded autologous monocytes for raising alloreactive CD4+ T cells. The autologous monocytes loaded with dimers (pulsed the specific peptide) were served as stimulating cells. PBLs obtained by density gradient centrifugation were served as stimulated cells. Here an allogeneic MHC classⅡmolecule associated with its restricted peptide is attached to monocytes. The DR15 dimer-loaded monocytes "present" the allogeneic epitope and induce autologous PBLs to generate peptide-specific allogeneic CD4+ T cells.The results of'CFSE dilution assay in vitro show that the dimer-loaded monocyte is able to stimulate autologous CD4+ T-cell proliferation; and specific dimer staining and ELISPOT assays reveal the allogeneic CD4+ T cells are pMHC specific.3. The tumor antigen peptide-specific allogeneic CD4+ T cells can be efficient at tumor rejection in vivoThe DR15-ve PBLs were stimulated by two cycles of tumor peptide/DR15 dimer-loaded autologous monocytes for 14 d, and the CD4+ T cells purified from the bulk were adoptively transferred into nude mice challenged by SiHa cells. The result showed that the percent of tumor free mice was significantly higher in the specific group than in control groups and the percent survival of mice was also significantly higher in the specific group than in control groups. The result of immunohistochemistry showed that there were much more CD4+ cells infiltrating into tumors in the specific group than in control groups.In conclusion, peptide-specific allogeneic CD4+ T cells are induced by cocultruing DR15-ve PBLs and peptide/DR15 dimer-loaded autologous monocytes in this study and the CD4+ T cells have specific biological function in vitro and in vivo. Tumor antigen peptide-specific allogeneic CD4+ T cells infiltrate obviously into the tumor tissue of nude mice and inhibit the growth of the tumor, which verifies alloreactive CD4+ T cells play an important role in GVTR and support the development of clinical strategies focusing on exploiting the function of tumor-reactive CD4+ T cells. This study also affords a new strategy preparing allogenic tumor antigen-specific CD4+ T cells.