Laboratory of Experimental Hematology, Vaccine & Infectious Disease Institute (Vaxinfectio), University of Antwerp, Antwerp University Hospital, Wilrijkstraat 10, B-2650 Edegem, Belgium.
J Immunol Methods. 2010 May 31;357(1-2):51-4. doi: 10.1016/j.jim.2010.03.001. Epub 2010 Mar 6.
Interferon (IFN)-gamma ELISPOT can be used to monitor the magnitude of virus-specific cellular immune responses in vaccine trials. Often, IFN-gamma ELISPOT is performed with cryopreserved peripheral blood mononuclear cells (PBMC). However, it has not been well defined yet to what extent diminished cell viability of PBMC following cryopreservation affects IFN-gamma responses in ELISPOT. Therefore, we assessed the influence of apoptotic cells on the number of spot-forming cells (SFC) in IFN-gamma ELISPOT using a gradient of UV-irradiated apoptotic PBMC and viral antigens derived from varicella zoster virus (VZV) and cytomegalovirus (CMV). No SFC were observed when UV-irradiated apoptotic cells were stimulated with VZV or CMV antigens. Moreover, presence of apoptotic cells among viable T cells hampered the detection of SFC following stimulation with VZV or CMV cell lysates, but not with CMVpp65 peptide pool. Statistical analysis showed that mainly late apoptotic cells, staining both Annexin V and 7-amino-actinomycin D (7-AAD), were associated with a decreased number of SFC. In conclusion, it is recommended to use highly viable thawed PBMC for the detection of virus-specific cellular immune responses by IFN-gamma ELISPOT, since the detection of CMV- and VZV-specific T cell responses stimulated by cell lysates was significantly impeded by the presence of apoptotic cells.
干扰素 (IFN)-γ ELISPOT 可用于监测疫苗试验中病毒特异性细胞免疫应答的程度。通常，使用冷冻保存的外周血单核细胞 (PBMC) 进行 IFN-γ ELISPOT。然而，冷冻保存后 PBMC 细胞活力降低对 ELISPOT 中 IFN-γ 反应的影响程度尚未得到很好的定义。因此，我们使用梯度紫外线照射的凋亡 PBMC 和源自水痘带状疱疹病毒 (VZV) 和巨细胞病毒 (CMV) 的病毒抗原，评估了凋亡细胞对 IFN-γ ELISPOT 中斑点形成细胞 (SFC) 数量的影响。用 VZV 或 CMV 抗原刺激紫外线照射的凋亡细胞时，未观察到 SFC。此外，在活 T 细胞中存在凋亡细胞会阻碍 VZV 或 CMV 细胞裂解物刺激后 SFC 的检测，但不阻碍 CMVpp65 肽库的检测。统计学分析表明，主要是晚期凋亡细胞，同时染色 Annexin V 和 7-氨基放线菌素 D (7-AAD)，与 SFC 数量减少相关。总之，建议使用高活力的解冻 PBMC 进行 IFN-γ ELISPOT 检测病毒特异性细胞免疫应答，因为细胞裂解物刺激的 CMV 和 VZV 特异性 T 细胞反应的检测受到凋亡细胞的显著阻碍。
