Department of Pathobiology & Diagnostic Investigation, Children's Hospital Boston, East Lansing, Michigan, USA.
Department of Pharmacology & Toxicology, Michigan State University, East Lansing, Michigan, USA.
Am J Reprod Immunol. 2022 Nov;88(5):e13614. doi: 10.1111/aji.13614. Epub 2022 Sep 11.
Leukocytes from the maternal-fetal interface are a valuable tool to study local changes in immune function during pregnancy; however, sampling can be challenging due to inadequate tissue availability and the invasive nature of placental bed biopsy. Here, we aim to purify and characterize leukocytes from paired peripheral and uterine blood samples to assess whether a less invasive method of uterine blood collection could yield a population of enriched uterine leukocytes suitable for ex vivo and in vitro analyses.
Human peripheral blood mononuclear cells (PBMC) and uterine blood mononuclear cells (UBMC) expressed from surgical gauze post C-section were isolated, and immunophenotypic information was acquired by multi-parameter flow cytometry. PBMC and UBMC were stained for markers used to define T and B lymphocytes, macrophages, regulatory T (T ) cells, and natural killer (NK) cells. Prime flow was performed to check expression and analysis of CD16 CD56 and CD16 CD56++ NK transcripts in PBMC and UBMC samples.
Immunophenotyping revealed that over 95% of both live PBMC and UBMC consisted of CD45 leukocytes. Higher percentages of CD16 CD56 , characterized as uterine NK (uNK) cells, were observed in UBMC samples as compared to PBMC samples (18.41% of CD45 CD3 vs. 2.73%, respectively), suggesting that CD16 CD56 cells were enriched in these samples. In UBMC, 49.64% of CD3-negative cells were of peripheral NK phenotype (CD16 CD56 ), suggesting infiltration of maternal peripheral NK (pNK) cell in the uterine interface.
Intrauterine leukocytes, especially CD16 CD56 NK cells, can be collected in sufficient numbers with increased purity by sampling the uterine cavity postdelivery with surgical gauze. Our results suggest that this non-invasive protocol is a useful sampling technique for isolating CD16 CD56 cells, however, due to peripheral blood contamination, the NK cell yield could be lower compared to actual decidual or endometrial samples post-partum which is more invasive.
来自母胎界面的白细胞是研究妊娠期间局部免疫功能变化的有价值工具；然而，由于组织可用性不足和胎盘床活检的侵入性，采样具有挑战性。在这里，我们旨在从配对的外周血和子宫血样本中纯化和表征白细胞，以评估一种侵入性较小的子宫血采集方法是否可以产生适合离体和体外分析的富含子宫白细胞的群体。
从剖宫产后的手术纱布中分离出人类外周血单核细胞（PBMC）和子宫血单核细胞（UBMC），并通过多参数流式细胞术获得免疫表型信息。对 PBMC 和 UBMC 进行染色，以定义 T 和 B 淋巴细胞、巨噬细胞、调节性 T（Treg）细胞和自然杀伤（NK）细胞的标志物。进行初步流式细胞术检查以检查 CD16+CD56+和 CD16+CD56++NK 转录本在 PBMC 和 UBMC 样本中的表达和分析。
免疫表型分析显示，活 PBMC 和 UBMC 中超过 95%由 CD45 白细胞组成。与 PBMC 样本相比，UBMC 样本中观察到更高比例的 CD16+CD56+，其特征为子宫 NK（uNK）细胞（CD45+CD3-，分别为 18.41%和 2.73%），提示 CD16+CD56+细胞在这些样本中得到了富集。在 UBMC 中，49.64%的 CD3-细胞为外周 NK 表型（CD16+CD56+），提示母体外周 NK（pNK）细胞浸润到子宫界面。
通过用手术纱布在产后采集子宫腔，可以以增加的纯度收集足够数量的宫内白细胞，特别是 CD16+CD56+NK 细胞。我们的结果表明，这种非侵入性方案是一种有用的采样技术，用于分离 CD16+CD56+细胞，但是，由于外周血污染，与产后更具侵入性的实际蜕膜或子宫内膜样本相比，NK 细胞的产量可能较低。
