| Cancer has become a major threat to human life and health.At present,conventional treatment methods include surgery,radiotherapy,chemotherapy and adoptive immunotherapy,but the clinical treatment effect is not ideal.Adoptive immunotherapy is gradually becoming a research hotspot in the field of malignant tumors because of its good curative effect and less toxic and side effects.Obtaining effector T cells sensitized to tumor cells is the key factor to determine the effect of adoptive immunotherapy.Tumor draining lymph node(Tumor-draining lymph node,TDLN)is a lymph node located in the tumor drainage area.Because tdln is in a special position in the body,dendritic cells(Dendritic cell,DC)containing information related to tumor antigen will be produced after long-term stimulation of tumor cell antigen and a large number of immune cells sensitized to tumor,such as T lymphocytes,B lymphocytes and natural killer cells(Natural killer cell,NK),so TDLN is a potential way to obtain immune cells sensitized to tumor cells more conveniently.Chimeric antigen receptor-T cell(Chimeric antigen receptor-T cell,CAR-T)refers to a class of T cells that can recognize specific target antigens in MHC unrestricted manner after gene modification and continuously expand activated T cells.After car transformation,T cells are given human leucocyte antigen(Human leukocyte antigen,HLA)the ability to target and recognize tumor associated antigens in an independent manner to resist and eliminate tumors.CAR-T is compared with T cell receptor(T cell receptor,TCR)in natural resting state it can more specifically and accurately identify specific targets.With the in-depth expansion,improvement and optimization of CAR-T exploration,this therapy will eventually benefit cancer patients to a great extent and is expected to overcome solid tumors.Objective:Based on CAR-T adoptive immunotherapy,T cells sensitized to autologous tumor cells in TDLN were modified with chimeric antigen receptor to explore the establishment of chimeric antigen receptor immune cells with the advantage of sensitization to autologous tumor cells and their antitumor activity in vitro.Methods:Lentivirus Expression Vector LentiV3-Luc-mCD19 over-expressing luciferase and mouse CD 19 antigen was constructed by molecular cloning technology,and 4T1-Luc-mCD19 cell line stably transformed luciferase and mouse CD 19 antigen was obtained by lentivirus packaging system;After subcutaneous tumor bearing BALB/c mice,TDLN was removed from axillary drainage lymph nodes,and TDLN-T cells were separated by CD4+/CD8+magnetic beads;The resting spleen-T was obtained by magnetic bead sorting from the spleen of normal mice,and the immunophenotype was detected by flow cytometry.At the same time,a reverse transcription expression vector pMIG-mCD19scFv-CAR containing the secondgeneration CAR structure of mCD19scFv was constructed.spleen-CAR-T cells and TDLN-CAR-T cells targeting mouse CD 19 were obtained by retroviral packaging and concentration.The transfection efficiency was detected by flow cytometry.The killing rates of spleen-T cells,TDLN-T cells,spleen-CAR-T cells and TDLN-CAR-T cells were detected according to different effect target ratios,and the IFN-γ and IL-2 in the co-culture supernatant was detected by ELISA.Results:(1)lentiviral expression vector LentiV3-Luc-mCD19 and retroviral expression vector pMIG-mCD19scFv-CAR were successfully constructed.(2)4T1-Luc-mCD19 cell line stably transformed with luciferase and mouse CD 19 antigen was successfully screened,and a mouse solid tumor model was established to obtain TDLN.(3)spleen-T cells in resting state and TDLN-T cells sensitized to tumor were successfully isolated from mice.spleen-CAR-T and TDLN-CAR-T cells were obtained by retroviral transfection.T cell immunophenotype and transfection efficiency were detected by flow cytometry.(4)The results of co-culture showed that the killing efficiency of TDLN-T on autologous tumor cells was higher than that of spleen-T,and the killing effect of CAR-T cells derived from TDLN-T cells was higher than that of CAR-T cells in natural state.Conclusion:a large number of immune cells can be obtained in TDLN of tumor bearing mice.Compared with spleen-T cells of normal mice,TDLN-T has better recognition and killing effect on autologous tumor cells,and it can show better antitumor activity after modified by chimeric antigen receptor than CAR-T cells in resting state. |
