Ye Wei, Xing Yun, Paustian Christopher, van de Ven Rieneke, Moudgil Tarsem, Hilton Traci L, Fox Bernard A, Urba Walter J, Zhao Wei, Hu Hong-Ming
Medical School, Southeast University, 87 Dingjiaqiao Street, 210009 Nanjing, Jiangsu, PR China.
J Transl Med. 2014 Apr 16;12:100. doi: 10.1186/1479-5876-12-100.
Autophagy regulates innate and adaptive immune responses to pathogens and tumors. We have reported that autophagosomes derived from tumor cells after proteasome inhibition, DRibbles (Defective ribosomal products in blebs), were excellent sources of antigens for efficient cross priming of tumor-specific CD8⁺ T cells, which mediated regression of established tumors in mice. But the activity of DRibbles in human has not been reported.
DRibbles or cell lysates derived from HEK293T or UbiLT3 cell lines expressing cytomegalovirus (CMV) pp65 protein or transfected with a plasmid encoding dominant HLA-A2 restricted CMV, Epstein-Barr virus (EBV), and Influenza (Flu) epitopes (CEF) were loaded onto human monocytes or PBMCs and the response of human CMV pp65 or CEF antigen-specific CD4⁺ and CD8⁺ memory T cells was detected by intracellular staining. The effect of cytokines (GM-CSF, IL-4, IL-12, TNF-α, IFN-α and IFN-γ) TLR agonists (Lipopolysaccharide, Polyinosinic-polycytidylic acid (poly(I:C), M52-CpG, R848, TLR2 ligand) and CD40 ligand on the cross-presentation of antigens contained in DRibbles or cell lysates was explored.
In this study we showed that purified monocytes, or human PBMCs, loaded with DRibbles isolated from cells expressing CMV or CEF epitopes, could activate CMV- or CEF-specific memory T cells. DRibbles were significantly more efficient at stimulating CD8⁺ memory T cells compared to cell lysates expressing the same antigenic epitopes. We optimized the conditions for T-cell activation and IFN-γ production following direct loading of DRibbles onto PBMCs. We found that the addition of Poly(I:C), CD40 ligand, and GM-CSF to the PBMCs together with DRibbles significantly increased the level of CD8⁺ T cell responses.
DRibbles containing specific viral antigens are an efficient ex vivo activator of human antigen-specific memory T cells specific for those antigens. This function could be enhanced by combining with Poly(I:C), CD40 ligand, and GM-CSF. This study provides proof-of-concept for applying this strategy to activate memory T cells against other antigens, including tumor-specific T cells ex vivo for immunological monitoring and adoptive immunotherapy, and in vivo as vaccines for patients with cancer.
自噬调节机体对病原体和肿瘤的先天性及适应性免疫反应。我们曾报道，蛋白酶体抑制后肿瘤细胞衍生的自噬体，即“泡状核糖体产物缺陷（DRibbles）”，是高效交叉启动肿瘤特异性CD8⁺T细胞的优质抗原来源，这些CD8⁺T细胞介导了小鼠体内已形成肿瘤的消退。但DRibbles在人体中的活性尚未见报道。
将从表达巨细胞病毒（CMV）pp65蛋白的HEK293T或UbiLT3细胞系中分离得到的DRibbles或细胞裂解物，或用编码显性HLA - A2限制性CMV、爱泼斯坦 - 巴尔病毒（EBV）和流感（Flu）表位（CEF）的质粒转染后的细胞裂解物，负载到人单核细胞或外周血单个核细胞（PBMCs）上，通过细胞内染色检测人CMV pp65或CEF抗原特异性CD4⁺和CD8⁺记忆T细胞的反应。探讨细胞因子（粒细胞 - 巨噬细胞集落刺激因子（GM - CSF）、白细胞介素 - 4（IL - 4）、白细胞介素 - 12（IL - 12）、肿瘤坏死因子 - α（TNF - α）、干扰素 - α（IFN - α）和干扰素 - γ（IFN - γ））、Toll样受体（TLR）激动剂（脂多糖、聚肌苷酸 - 聚胞苷酸（聚（I:C））、M52 - CpG、R848、TLR2配体）和CD40配体对DRibbles或细胞裂解物中所含抗原交叉呈递的影响。
在本研究中，我们发现用从表达CMV或CEF表位的细胞中分离得到的DRibbles负载的纯化单核细胞或人PBMCs，能够激活CMV或CEF特异性记忆T细胞。与表达相同抗原表位的细胞裂解物相比，DRibbles在刺激CD8⁺记忆T细胞方面效率显著更高。我们优化了将DRibbles直接负载到PBMCs上后T细胞激活和IFN - γ产生的条件。我们发现，在PBMCs中加入聚（I:C）、CD40配体和GM - CSF以及DRibbles，可显著提高CD8⁺T细胞反应水平。
含有特定病毒抗原的DRibbles是体外激活针对这些抗原的人抗原特异性记忆T细胞的有效激活剂。通过与聚（I:C）、CD40配体和GM - CSF联合使用，这一功能可得到增强。本研究为将该策略应用于激活针对其他抗原的记忆T细胞提供了概念验证，包括体外激活肿瘤特异性T细胞用于免疫监测和过继性免疫治疗，以及体内作为癌症患者的疫苗。
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