Effects of cryopreservation on effector cells for antibody dependent cell-mediated cytotoxicity (ADCC) and natural killer (NK) cell activity in (51)Cr-release and CD107a assays.
Effects of cryopreservation on effector cells for antibody dependent cell-mediated cytotoxicity (ADCC) and natural killer (NK) cell activity in (51)Cr-release and CD107a assays.
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DOI：
10.1016/j.jim.2014.01.017
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发表时间：
2014-04
影响因子：
2.2
通讯作者：
Baum LL
中科院分区：
文献类型：
--
作者：
Mata MM;Mahmood F;Sowell RT;Baum LL
新鲜分离的PBMC在评估抗体依赖细胞介导的细胞毒性(ADCC)和NK活性的功能分析中被广泛用作效应细胞；然而，它们引入了自然-个体供体对供体的差异性。在细胞毒性试验中，冷冻保存的PBMC提供了比新鲜细胞更一致的效应源。我们的目的是确定冻存效应PBMC对细胞频率的影响，以及对ADCC和NK活性的大小和特异性的影响。以新鲜、冷冻/隔夜静息和冷冻/未静息的PBMC为效应细胞，进行51Cr释放和CD107a脱颗粒实验。冻存/过夜休眠PBMC的ADCC和NK活性均高于新鲜PBMC，但冻存/未休眠PBMC的ADCC和NK活性显著低于新鲜PBMC。背景：与新鲜的PBMC或冷冻过夜和静息的PBMC相比，在没有靶细胞刺激的情况下，冷冻/未休息的PBMC中CD107a脱颗粒更多。冻存过夜的PBMC中CD16+CD56dim NK细胞和CD14+单核细胞的百分率低于新鲜PBMC。CD56dim NK细胞表面CD16的表达在所有PBMC处理组中相似。冻存过夜的PBMC与新鲜的PBMC效应器相当。在用51Cr释放法或CD107a脱颗粒法评估ADCC或NK活性时，冷冻并立即使用的PBMC的活性最低。对介导ADCC的抗体的临床研究将受益于使用在化验前已冷冻、解冻和休息一夜的效应细胞。
Freshly isolated PBMC are broadly used as effector cells in functional assays that evaluate antibody-dependent cell mediated cytotoxicity (ADCC) and NK activity; however, they introduce natural-individual donor-to-donor variability. Cryopreserved PBMC provide a more consistent source of effectors than fresh cells in cytotoxicity assays. Our objective was to determine the effects of cryopreservation of effector PBMC on cell frequency, and on the magnitude and specificity of ADCC and NK activity. Fresh, frozen/overnight rested and frozen/not rested PBMC were used as effector cells in 51Cr-release and CD107a degranulation assays. Frozen/overnight rested PBMC had higher ADCC and NK activity in both assays when compared to fresh PBMC; however, when using frozen/not rested PBMC, ADCC and NK activities were significantly lower than fresh PBMC. Background CD107a degranulation in the absence of target cell stimulation was greater in PBMC that were frozen/not rested when compared to fresh PBMC or PBMC that were frozen overnight and rested. The percentages of CD16+CD56dim NK cells and CD14+ monocytes were lower in PBMC that were frozen and rested overnight than in fresh PBMC. CD16 expression on CD56dim NK cells was similar for all PBMC treatments. PBMC that were frozen and rested overnight were comparable to fresh PBMC effectors. PBMC that were frozen and used immediately when evaluating ADCC or NK activity using either a 51Cr-release assay or a CD107a degranulation assay had the lowest activity. Clinical studies of antibodies that mediate ADCC would benefit from using effector cells that have been frozen, thawed and rested overnight prior to assay.
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