ABSTRACT
@#[摘 要] 目的：通过构建表达IL-12的小鼠CAR-T细胞，探讨经尾静脉将其输注于小鼠体内建立细胞因子释放综合征（CRS）模型的方法。方法：构建基于靶向鼠源CD19的CAR分子，包装逆转录病毒载体并感染小鼠T细胞构建mCD19-CAR-T、mCD19/IL-12-CAR-T细胞。通过构建小鼠体内胰腺癌Panc02-CD19细胞移植瘤模型，检测mCD19/IL-12-CAR-T细胞的抗肿瘤活性，ELISA法检测两种CAR-T细胞IL-12和IFN-γ分泌水平；经小鼠尾静脉输注mCD19/IL-12-CAR-T 细胞构建CAR-T细胞CRS小鼠模型，流式细胞术检测小鼠血清中IL-6、MCP-1、IL-1、IL-10、TNF-α、IFN-γ等细胞因子的含量，H-E染色法观察荷瘤小鼠肝、脾、肺和肾的病理组织学变化。结果：经过培养扩增的mCD19/IL-12-CAR-T细胞能有效分泌IL-12，CAR阳性率达（56.9±5.4）%；与非靶细胞Panc02或靶细胞Panc02-CD19共培养时，均能高分泌IFN-γ。成功构建小鼠胰腺癌Panc02-CD19细胞移植瘤模型，经小鼠尾静脉注射1×106个mCD19/IL-12-CAR-T细胞能显著抑制移植瘤的生长，但未能诱发严重CRS；输注2×106个mCD19/IL-12-CAR-T细胞后，小鼠出现体质量减轻、血清炎性因子水平升高、组织损伤，最终导致死亡等一系列典型CRS表现。结论：成功构建IL-12-CAR-T细胞诱发的小鼠CRS模型，其稳定性好、重复性高，具有广泛的应用前景。
ABSTRACT
@# Objective: To investigate the expression and clinical significance of PD-1 molecule in tumor cells (T-ALL cells) derived from the patient with T-cell acute lymphoblastic leukemia (T-ALL). Methods: T-ALL cells from one patient and PBMCs from four healthy volunteers provided by the Department of Hematology in Jiangsu Provincial Hospital of Traditional Chinese Medicine in December 2015, and human 293T/PD-1 cells provided by Persongen Bio Therapeutics (Suzhou) Co., Ltd. were used for this study. The mouse T-ALL xenograft model was constructed by injecting T-ALL cells into tail vein of B-NDG mice, and flow cytometry was used to verify whether the cells obtained from the spleen of transplanted mice were mainly consisted of T-ALL cells. Flow cytometry was used to study the protein expression of PD-1 in T-ALL cells, and RT-PCR was applied to further verify the mRNA expression of PD-1 in T-ALL cells. The PD-1 gene in T-ALL cells was sequenced by SNP genotyping to detect whether the DNA sequence of PD-1 gene changed. PD-1 inhibitor was used in vitro to study their effects on proliferation, apoptosis, and the mRNA expression levels of related factors in T-ALL cells. Results: The mouse T-ALL xenograft model was successfully constructed and verified by flow cytometry as T-ALL. PD-1 was highly expressed at both mRNA and protein levels in T-ALL cells (all P<0.01). A C-to-T mutation was detected in the fifth exon of the PD-1 gene. PD-1 inhibitor had no significant effect on proliferation and apoptosis of T-ALL cells in vitro; PD-1 inhibitor up-regulated the mRNA expression of tumor-suppressor protein IGFBP3 and decreased the mRNA expression of oncoprotein SULT1A3 (all P<0.01). Conclusion: PD-1 is highly expressed in T-ALL cells, and PD-1 could be used as a target for clinical diagnosis and treatment for T-ALL.
ABSTRACT
@#Objective:To investigate the tumor-specific neoantigen for primary plasma cell leukemia (PCL) using gene sequencing technology combined with bioinformatic analysis. Methods: Peripheral blood samples of one patient with primary PCL during relapse and remission periods were collected. HLA molecular typing was performed using polymerase chain reaction with sequencing-based typing; whole-exome and transcriptome were sequenced by next-generation sequencing method; and bioinformatics software NetMHCpan was used to predict neoantigens. Results: Six tumor-specific missense mutations were found in the patient's peripheral blood during relapse period, located in genes FRG1, MLL3, SVIL, MYOM1, ZDHHC11 and RFPL4A.Considering patient's HLA sub-types, 43 neoantigens were predicted via bioinformatics. Considering that FRG1 and MLL3 had relatively high gene expression levels, 20 neoantigens derived from mutations of the two genes were preferentially selected, among which four neoantigens had high affinity with the patient's HLA molecules and thus had potential clinical application value. Conclusion: The study has completed a tumor neoantigen screen and prediction for primary PCL. This practice demonstrates that predicting neoantigen based on tumor-specific somatic mutation is feasible for primary PCL.
ABSTRACT
@# Objective： ：To explore a novel chimeric antigen receptor (CAR)-T cell treatment to treat Multiple Myeloma (MM) via target B cell maturation antigen (BCMA). Methods： ：A CAR-BCMA molecular was constructed based on mouse originated BCMA scFv, and was packaged into lentiviral vector and transfected into T cells from healthy donors to construct CAR-BCMA-T cells. The BCMApositive cell lines A549-BCMA, A549-BCMAOFP and K562-BCMA were constructed as target cells. Then, the CAR-BCMA-T cells were co-incubated with the constructed target cells and human myeloma U266 cells, and the cytotoxic effects of CAR-BCMA-T cells were evaluated via CCK-8 and FACS. Finally, the CAR-BCMA-T cells originated from MM patients were constructed, and its cytotoxicity against A549-BCMA were examined; in addition, the IFN-γ release level in CAR-BCMA-T cells was evaluated by ELISA and FACS. Results: After 11 days’incubation, the CAR-BCMA-T cells originated from healthy donors amplified 300 times with a positive rate of 43%. The BCMApositive target cell lines were constructed successfully. Under an effector : target ratio of 5:1, the killing rates of CARBCMA-T cells against A549-BCMA, K562-BCMA and U266 were about 80%, 60%, and 80%, respectively, which were significantly higher than those against BCMA negative cells; and the cytotoxicity was related to the BCMA expression level in target cells. What’ s more, at the effector : target ratio of 20:1, the CAR-BCMA-T cells originated from MM patients were demonstrated to exhibit a killing rate of more than 95% againstA549-BCMApositive cells, and produced large amount of IFN-γ. Conclusion: CAR-BCMA-T cells originated from both healthy and MM donors were successfully constructed, and they can effectively and specifically kill BCMA positive tumor cells.
ABSTRACT
@#[Abstract] Objective: To establish a chimeric antigen receptor（CAR）modified T cells specifically targeting CD19 molecule (CD19CAR-T cells) and to testify their in vitro killing effect on target cells. Methods: CD19-CAR fragments yielded by PCR were constructed into pCDH-GFP lentiviral vectors by molecular cloning technology. The packaged lentiviral particles were transducted into CD3+ T cells of donors. Transduction efficiency was measured by flow cytometry and PCR. The in vitro cytotoxicity of obtained CD19CAR-T cells against CD19+ Ramos cells was tested by 7-AAD staining. Results: The amplification folds of CD3+ T cells increased to (78.8± 23.2) folds after in vitro culture for 10 days, and about (58.3±5.4)% cells expressing GFP.About (57.4±9.3)% CD19+Ramos cells were specifically killed by the CD19-CAR-T cells in vitro at the E∶T ratio of 5∶1. Conclusion: This study successfully established an effective method for constructing and amplifying CD19-CAR-T cells in vitro, which showed profound efficiency and specific cytotoxity against CD19+ Ramos cells.And this report might provide an experimental evidence for clinical treatment of CD19+ B cell neoplasmas.