外周血干细胞采集物来源的巨细胞病毒特异性T细胞的体外培养和扩增

Culture and expansion of CMV-specific T cells derived from peripheral blood stem cells in vitro

目的建立巨细胞病毒特异性细胞毒性T淋巴细胞(CMV CTL)体外扩增的方法。方法用1μg/mL全长巨细胞病毒pp65(CMV pp65)多肽体外多轮刺激由粒细胞-集落刺激因子(G-CSF)动员的外周血干细胞采集物中分离的单个核细胞(PBMC),同时加入白细胞介素2(IL-2)、 IL-15、 IL-21扩增20 d。在培养第7天,加入丝裂霉素处理的负载CMV pp65抗原肽的自体PBMC,第10天补充经γ射线辐照处理并负载CMV pp65抗原肽的PBMC和CD3(OKT3);对照组为未加入抗原肽进行扩增的PBMC组。采用多色流式细胞术分别对扩增前、扩增后的T淋巴细胞表型及细胞内肿瘤坏死因子α(TNF-α)和γ干扰素(IFN-γ)的分泌水平进行分析, ELISA检测供者血清中CMV IgM/IgG抗体滴度水平。结果培养后可以收集得到(165.26±6.14)×10~6个细胞,其中CD3^+ T细胞占89.21%, CD8^+ T细胞占CD3^+ T细胞的(43.54±28.03)%, CD4^+ T细胞占CD3^+ T细胞(34.23±26.18)%。获得的CD3^+ T细胞以效应记忆性T细胞(T_(EM))为主,且扩增培养后的CD8^+和CD4^+ T_(EM)比例较培养前显著增高。另外,培养后干细胞样记忆性T细胞(T_(SCM))和组织原位记忆T细胞(T_(RM))的比例均较培养前显著增加。在功能实验中,培养后得到的IFN-γ^+的分泌型CMV特异性CD8^+ T细胞群比例较扩增前明显增加, TNF-α^+ CMV特异性CD8^+ T细胞比例呈增长趋势;能够分别分泌IFN-γ和TNF-α的CMV特异性CD4^+ T细胞群比例与培养前无明显变化。此外,供者体内CMV IgG水平与供者年龄呈现正相关,且在该培养体系下, IFN-γ^+和TNF-α^+ CMV特异性T细胞扩增比例与供者年龄呈负相关。结论本研究成功建立了在体外有效的培养和扩增CMV CTL的方法。

Objective To establish a method for in vitro expansion of cytomegalovirus-specific cytotoxic T lymphocytes(CMV CTL).Methods Full-length cytomegalovirus pp65(CMV pp65)polypeptide 1μg/mL was used to repeat rounds of in vitro stimulation of peripheral blood mononuclear cells(PBMCs)isolated from peripheral blood stem cell collections mobilized by granulocyte-colony stimulating factor(G-CSF),and interleukin 2(IL-2),IL-15 and IL-21 were also added for amplification for 20 days.On the 7th day of culture,autologous PBMCs loaded with CMV pp65 antigen peptide were added after being processed by mitomycin.On day 10,γray-radiated PBMCs loaded with CMV pp65 antigen peptide and CD3(OKT3)were supplemented.For the control group,no antigen was added into PBMCs for amplification.The phenotypes of T lymphocytes and the secretion levels of tumor necrosis factor alpha(TNF-α)and interferon gamma(IFN-γ)before and after amplification were analyzed by polychromatic flow cytometry.The titer of serum CMV IgM/IgG antibody of the donors was detected by ELISA.Results(165.26±6.14)×106 cells were harvested after culture,including CD3+T cells 89.21%.CD8+T cells accounted for(43.54±28.03)%of CD3+T cells,and CD4+T cells for(34.23±26.18)%.The CD3+T cells obtained were predominated by effector memory T cells(TEM),and the proportions of TEM in CD8+T cells and CD4+T cells after amplification culture were significantly higher than those before culture.Additionally,the proportions of stem cell-like memory T cells(TSCM)and tissue resident memory T cells(TRM)after culture were obviously raised than those before culture.In the functional experiment,the proportion of IFN-γ+secreting CMV-specific CD8+T cells after amplification was much higher than that before amplification,and the proportion of TNF-α+CMV-specific CD8+T cells was on the rise;the proportions of CMV-specific CD4+T cells that could secrete IFN-γand TNF-αhad no obvious changes.Furthermore,the level of CMV IgG was positively correlated with the donor’s ages,and in such a culture system,the proportions of amplified IFN-γ+and TNF-α+secreting CMV-specific T cells were negatively correlated with the donor’s ages.Conclusion We have successfully established an effective method to amplify CMV CTL in vitro.