论著 · 基础研究

B7基因修饰的白血病细胞外泌体的抗白血病效应

  • 张涤凡 ,
  • 王明慧 ,
  • 赵洁 ,
  • 万江波 ,
  • 黄方 ,
  • 郝思国
展开
  • 上海交通大学医学院附属新华医院血液科,上海 200092
张涤凡(1998—),女,住院医师,硕士;电子信箱:zhangdifan121@163.com
郝思国,主任医师,博士;电子信箱:haosiguo@xinhuamed.com.cn
黄 方,主治医师,博士;电子信箱:huangfang335@163.com

收稿日期: 2024-05-20

  录用日期: 2024-10-14

  网络出版日期: 2025-02-28

基金资助

国家自然科学基金(81470314)

Anti-leukemia effect of B7 gene-modified leukemia cell-derived exosomes

  • ZHANG Difan ,
  • WANG Minghui ,
  • ZHAO Jie ,
  • WAN Jiangbo ,
  • HUANG Fang ,
  • HAO Siguo
Expand
  • Department of Hematology, Xinhua Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200092, China
HAO Siguo, E-mail: haosiguo@xinhuamed.com.cn
HUANG Fang, E-mail: huangfang335@163.com.

Received date: 2024-05-20

  Accepted date: 2024-10-14

  Online published: 2025-02-28

Supported by

National Natural Science Foundation of China(81470314)

摘要

目的·探究高表达B7分子(共刺激分子CD80和CD86)的白血病细胞外泌体在荷瘤小鼠中的免疫治疗效应。方法·L1210白血病细胞株转染含有CD80CD86基因的慢病毒载体,从而获得高表达CD80和CD86分子的白血病细胞,分离其培养上清液获得高表达CD80和CD86分子的外泌体LEX-8086。应用透射电子显微镜(电镜)和Western blotting分析LEX-8086的生物学特征。建立DBA/2-白血病荷瘤小鼠模型,将荷瘤小鼠分为LEX-8086组和PBS组,LEX-8086组分别在肿瘤细胞接种后7 d和12 d尾静脉注射LEX-8086 500 μL(150 μg/mL),PBS组同时注射等体积PBS;观察2组小鼠的体质量、肿瘤体积、肿瘤质量及生存期差异。应用流式细胞术分析2组小鼠脾脏和淋巴结免疫细胞的变化特点。应用Western blotting和实时荧光定量PCR(qPCR)检测2组小鼠脾脏M1型和M2型巨噬细胞标志物的表达情况。结果·从高表达CD80和CD86分子的L1210细胞上清液中分离获得LEX-8086,透射电镜显示为直径约100 nm的盘状囊泡结构,Western blotting结果显示其表达外泌体特异性标志物。动物实验结果显示,LEX-8086组较PBS组,体质量无明显改变,肿瘤体积和肿瘤相对质量比均显著下降且生存期显著延长(均P<0.05)。流式细胞术结果显示,LEX-8086组淋巴结、脾脏内自然杀伤细胞比例、CD4+ T细胞比例均显著升高(均P<0.05),而CD8+ T细胞仅在淋巴结内显著升高(P=0.012)。此外,流式细胞术结果显示LEX-8086组小鼠脾脏中M1型巨噬细胞比例显著升高(P=0.003),M2型巨噬细胞比例无明显变化。Western blotting和qPCR同样显示,LEX-8086组小鼠脾脏中M1型巨噬细胞标志物白细胞介素-6和肿瘤坏死因子α mRNA和蛋白表达水平均显著升高(均P<0.05)。结论·共刺激分子CD80和CD86高表达的白血病细胞外泌体能够诱导一定的治疗性抗白血病效应,该效应可能是通过提高NK细胞、M1型巨噬细胞、CD4+ T细胞和CD8+ T细胞的比例实现的。

本文引用格式

张涤凡 , 王明慧 , 赵洁 , 万江波 , 黄方 , 郝思国 . B7基因修饰的白血病细胞外泌体的抗白血病效应[J]. 上海交通大学学报(医学版), 2025 , 45(2) : 129 -137 . DOI: 10.3969/j.issn.1674-8115.2025.02.001

Abstract

Objective ·To investigate the immunotherapeutic effects of exosomes derived from leukemia cells with high expression of B7 molecules (co-stimulatory molecules CD80 and CD86) in tumor-bearing mice. Methods ·L1210 leukemia cells were transfected with lentiviral vectors containing the CD80 and CD86 genes, and thereby the leukemia cells with high expression of CD80 and CD86 molecules were obtained. Exosomes LEX-8086 with high expression of CD80 and CD86 molecules were then isolated from the culture supernatant. The biological characteristics of LEX-8086 were analyzed by using transmission electron microscopy (TEM) and Western blotting. A DBA/2-leukemia tumor-bearing mouse model was established. The tumor-bearing mice were divided into LEX-8086 group and PBS group. The LEX-8086 group received intravenous injections of 500 μL LEX-8086 (150 μg/mL) via the tail vein 7 d and 12 d after tumor cell inoculation, while the PBS group received injections of equal volumes of PBS at the same time points. Differences in body weight, tumor volume, tumor mass, and survival of the mice between the two groups were observed. Flow cytometry was used to analyze the changes in the immune cells in spleens and lymph nodes of the mice in both groups. Western blotting and real-time fluorescent quantitative PCR (qPCR) were used to detect the expression of markers for M1 and M2 macrophages in the spleens of the mice in both groups. Results ·The exosomes LEX-8086 were isolated from the supernatant of L1210 cells overexpressing CD80 and CD86 molecules. TEM revealed that LEX-8086 exhibited a disc-shaped vesicular structure with a diameter of approximately 100 nm. Western blotting demonstrated the expression of exosome-specific markers. In the animal experiments, compared with the PBS group, the body weight of the LEX-8086 group showed no significant change, while both the tumor volume and the relative tumor mass ratio decreased significantly, and the survival was significantly prolonged (all P<0.05). Flow cytometry results indicated that the proportions of natural killer (NK) cells and CD4+ T cells in the lymph nodes and spleens of the LEX-8086 group significantly increased (all P<0.05); as for CD8+ T cells, they only increased in the lymph modes (P=0.012). Moreover, flow cytometry results showed that the proportion of M1 macrophages in the spleens of mice in the LEX-8086 group was significantly elevated (P=0.003), while the proportion of M2 macrophages remained unchanged. Western blotting and qPCR also revealed that the mRNA and protein expression levels of M1 macrophage markers interleukin-6 and tumor necrosis factor-α in the spleens of mice in the LEX-8086 group increased significantly (all P<0.05). Conclusion ·Exosomes derived from leukemia cells with high expression of co-stimulatory molecules CD80 and CD86 can induce a therapeutic anti-leukemic effect, which may be achieved by increasing the proportions of NK cells, M1 macrophages, CD4+ T cells, and CD8+ T cells.

参考文献

1 WHITESIDE T L. Tumor-derived exosomes and their role in cancer progression[J]. Adv Clin Chem, 2016, 74: 103-141.
2 SUN Z Y, XIE C Q, LIU H, et al. CD19 CAR-T cell therapy induced immunotherapy associated interstitial pneumonitis: a case report[J]. Front Immunol, 2022, 13: 778192.
3 RAJE N, BERDEJA J, LIN Y, et al. Anti-BCMA CAR T-cell therapy bb2121 in relapsed or refractory multiple myeloma[J]. N Engl J Med, 2019, 380(18): 1726-1737.
4 OTT P A, HU Z T, KESKIN D B, et al. An immunogenic personal neoantigen vaccine for patients with melanoma[J]. Nature, 2017, 547(7662): 217-221.
5 SHEIH A, VOILLET V, HANAFI L A, et al. Clonal kinetics and single-cell transcriptional profiling of CAR-T cells in patients undergoing CD19 CAR-T immunotherapy[J]. Nat Commun, 2020, 11(1): 219.
6 CHEN G, HUANG A C, ZHANG W, et al. Exosomal PD-L1 contributes to immunosuppression and is associated with anti-PD-1 response[J]. Nature, 2018, 560(7718): 382-386.
7 AHMAD A. Epigenetic regulation of immunosuppressive tumor-associated macrophages through dysregulated microRNAs[J]. Semin Cell Dev Biol, 2022, 124: 26-33.
8 NASERI M, BOZORGMEHR M, Z?LLER M, et al. Tumor-derived exosomes: the next generation of promising cell-free vaccines in cancer immunotherapy[J]. Oncoimmunology, 2020, 9(1): 1779991.
9 KRACKHARDT A M, HARIG S, WITZENS M, et al. T-cell responses against chronic lymphocytic leukemia cells: implications for immunotherapy[J]. Blood, 2002, 100(1): 167-173.
10 TOWNSEND S E, ALLISON J P. Tumor rejection after direct costimulation of CD8+ T cells by B7-transfected melanoma cells[J]. Science, 1993, 259(5093): 368-370.
11 CHENG Q Z, KANG Y, YAO B, et al. Genetically engineered-cell-membrane nanovesicles for cancer immunotherapy[J]. Adv Sci (Weinh), 2023, 10(26): e2302131.
12 HU W W, HUANG F, NING L X, et al. Enhanced immunogenicity of leukemia-derived exosomes via transfection with lentiviral vectors encoding costimulatory molecules[J]. Cell Oncol (Dordr), 2020, 43(5): 889-900.
13 LI J Q, HUANG F, JIANG Y, et al. A novel costimulatory molecule gene-modified leukemia cell-derived exosome-targeted CD4+ T cell vaccine efficiently enhances anti-leukemia immunity[J]. Front Immunol, 2022, 13: 1043484.
14 JOHNSON B D, YAN X C, SCHAUER D W, et al. Dual expression of CD80 and CD86 produces a tumor vaccine superior to single expression of either molecule[J]. Cell Immunol, 2003, 222(1): 15-26.
15 VASILEVKO V, GHOCHIKYAN A, HOLTERMAN M J, et al. CD80 (B7-1) and CD86 (B7-2) are functionally equivalent in the initiation and maintenance of CD4+ T-cell proliferation after activation with suboptimal doses of PHA[J]. DNA Cell Biol, 2002, 21(3): 137-149.
16 SIVORI S, PENDE D, QUATRINI L, et al. NK cells and ILCs in tumor immunotherapy[J]. Mol Aspects Med, 2021, 80: 100870.
17 TRACY S I, VENKATESH H, HEKIM C, et al. Combining nilotinib and PD-L1 blockade reverses CD4+ T-cell dysfunction and prevents relapse in acute B-cell leukemia[J]. Blood, 2022, 140(4): 335-348.
18 DISTLER E, ALBRECHT J, BRUNK A, et al. Patient-individualized CD8? cytolytic T-cell therapy effectively combats minimal residual leukemia in immunodeficient mice[J]. Int J Cancer, 2016, 138(5): 1256-1268.
文章导航

/