Establishment Of A Quantitative PCR Assay For Monitoring Chimeric Antigen Receptor T Cells In Peripheral Blood

Background:Chimeric Antigen Receptor T-Cell Immuno-therapy(CAR)is a newly synthesized artificial receptor that is replaced by a single chain variable fragment(scFv)of a tumor-specific monoclonal antibody.The variable regions of the alpha and beta chains of the T cell receptor(TCR)are formed by the attachment of scFv to co-stimulatory molecules(such as CD28 and 4-1BB,etc.)and the T cell signaling domain CD3(R).Chimeric antigen receptor T-cell immunotherapy(CAR-T)has achieved remarkable results in hematological neoplasm diseases and is considered to be one of the most promising approaches to treatment of cancer.Dynamic monitoring of the number of CAR-T cells in a patient can provide more detailed information about the patient's condition and predict the clinical effectiveness of CAR-T therapy.Therefore,it is necessary to develop a precise and simple method for detecting the number of CAR-T cells.Objectives:The purpose of this study was to develop a method for the detection of CAR-T cells in peripheral blood based on TaqMan real-time quantitative PCR(qPCR)for the monitoring of the number of CAR-T cells in peripheral blood after CAR-T treatment.Materials and Methods:Based on the TaqMan real-time fluorescence quantitative PCR method,specific CAR-T cell gene CAR and housekeeping gene GAPDH were detected to calculate the number of CAR-T cells and the proportion of peripheral blood nucleated cells.Specific steps include the extraction of peripheral blood cell genomic DNA using the following primers and probes,forward primer sequence:GGATTCGCCAGCCTCCAC,reverse primer sequence:AAACTTGGCTCTTGGAG TTGT and probe sequence:(FAM)-TCCCAGCCACTCCAGACCCTT-(MGB)in ABI VII7 fluorescence quantitative PCR The following reactions were performed on the instrument,40 cycles of denaturation at 95°C for 30 seconds and annealing at 60°C for30 seconds,during which fluorescence was measured.Results:This assay had a minimum detection limit of 1.2×10~1 copies/?l and a strong linear standard curve(y=-3.3682x+38.594,R~2=0.999)within the range of the input CAR gene(1.2×10~1 to 1.2×10~7 copies/?l).The reproducibility test showed a coefficient of variation ranging from 0.63%to 1.65%.Conclusion:TaqMan Real-Time PCR,which we have developed,is a method that can be used to detect CAR-T cells in peripheral blood,and it has the advantages of high sensitivity,high specificity and reproducibility.Objective: It was found that the expression of interferon-stimulated gene 15(Isg15)gene was increased in the testes of Sertoli cell specific Ar knockout(S-Ar-/y)mice.Objective: To confirm the effect of androgen receptor and its receptor on the expression of Isg15 gene in testicular tissue.Methods: The expression of Isg15 was detected by RT-q PCR and western blot.The distribution of Isg15 protein in mice testis was detected by immunohistochemistry and immunofluorescence.Results: In S-Ar-/y mice,the expression of Isg15 gene was significantly higher than that in wild-type mice,and the Isg15 protein was mainly expressed in spermatocytes.Conclusion: Androgen and its receptor signaling in mouse testis tissue support cells can inhibit the expression of interferon stimulator 15 gene in spermatocytes.