Strategy & Growth, Australian Red Cross Lifeblood, Sydney, NSW, Australia.
The Kirby Institute, University of NSW, Sydney, NSW, Australia.
Front Immunol. 2024 Aug 16;15:1382711. doi: 10.3389/fimmu.2024.1382711. eCollection 2024.
Many research laboratories have long-term repositories of cryopreserved peripheral blood mononuclear cells (PBMC), which are costly to maintain but are of uncertain utility for immunological studies after decades in storage. This study investigated preservation of cell surface phenotypes and functional capacity of PBMC from viraemic HIV+ patients and healthy seronegative control subjects, after more than 20 years of cryopreservation.
PBMC were assessed by 18-colour flow cytometry for major lymphocyte subsets within T, B, NK, and dendritic cells and monocytes. Markers of T-cell differentiation and activation were compared with original immunophenotyping performed in 1995/1996 on fresh blood at the time of collection. Functionality of PBMC was assessed by culture with influenza antigen or polyclonal T-cell activation, to measure upregulation of activation-induced CD25 and CD134 (OX40) on CD4 T cells and cytokine production at day 2, and proliferative CD25+ CD4 blasts at day 7. RNA was extracted from cultures containing proliferating CD4+ blast cells, and intracellular HIV RNA was measured using short amplicons for both the Double R and pol region pi code assays, whereas long 4-kbp amplicons were sequenced.
All major lymphocyte and T-cell subpopulations were conserved after long-term cryostorage, except for decreased proportions of activated CD38+HLA-DR+ CD4 and CD8 T cells in PBMC from HIV+ patients. Otherwise, differences in T-cell subpopulations between recent and long-term cryopreserved PBMC primarily reflected donor age-associated or HIV infection-associated effects on phenotypes. Proportions of naïve, memory, and effector subsets of T cells from thawed PBMC correlated with results from the original flow cytometric analysis of respective fresh blood samples. Antigen-specific and polyclonal T-cell responses were readily detected in cryopreserved PBMC from HIV+ patients and healthy control donors. Intracellular HIV RNA quantitation by pi code assay correlated with original plasma viral RNA load results. Full-length intracellular and supernatant-derived amplicons were generated from 5/12 donors, and sequences were ≥80% wild-type, consistent with replication competence.
This unique study provides strong rationale and validity for using well-maintained biorepositories to support immunovirological research even decades after collection.
许多研究实验室都有长期保存的冷冻外周血单核细胞（PBMC）储存库，这些细胞保存成本很高，但在储存数十年后，对于免疫研究的用途尚不确定。本研究调查了经过 20 多年冷冻保存后，来自病毒血症 HIV+患者和健康血清阴性对照者的 PBMC 的细胞表面表型和功能能力的保存情况。
通过 18 色流式细胞术评估 T、B、NK 和树突状细胞和单核细胞内的主要淋巴细胞亚群。与采集时新鲜血液在 1995/1996 年进行的原始免疫表型比较，比较 T 细胞分化和激活标志物。通过流感抗原或多克隆 T 细胞激活培养评估 PBMC 的功能，以测量 CD4 T 细胞上激活诱导的 CD25 和 CD134（OX40）的上调以及第 2 天的细胞因子产生，并在第 7 天测量增殖的 CD25+CD4 母细胞。从含有增殖 CD4+母细胞的培养物中提取 RNA，并使用针对 Double R 和 pol 区域 pi 码测定的短扩增子测量细胞内 HIV RNA，而 4-kbp 长扩增子则进行测序。
除了 HIV+患者的 PBMC 中激活的 CD38+HLA-DR+CD4 和 CD8 T 细胞比例降低外，所有主要的淋巴细胞和 T 细胞亚群在长期冷冻保存后都得到了保存。否则，近期和长期冷冻保存的 PBMC 之间 T 细胞亚群的差异主要反映了供体年龄相关或 HIV 感染相关的表型影响。解冻 PBMC 的 T 细胞幼稚、记忆和效应亚群的比例与相应新鲜血液样本的原始流式细胞分析结果相关。从 HIV+患者和健康对照供体的冷冻 PBMC 中容易检测到抗原特异性和多克隆 T 细胞反应。通过 pi 码测定进行细胞内 HIV RNA 定量与原始血浆病毒 RNA 负荷结果相关。从 12 名供体中的 5 名产生了全长细胞内和上清衍生扩增子，序列≥80%为野生型，与复制能力一致。
这项独特的研究为使用维护良好的生物库支持免疫病毒学研究提供了强有力的理由和有效性，即使在采集后几十年也是如此。
