Blood and Coagulation Research, US Army Institute of Surgical Research, JBSA Fort Sam Houston, TX, United States of America.
Blood and Coagulation Research, US Army Institute of Surgical Research, JBSA Fort Sam Houston, TX, United States of America; Department of Molecular Medicine, UT Health San Antonio, San Antonio, TX, United States of America.
J Immunol Methods. 2021 May;492:113000. doi: 10.1016/j.jim.2021.113000. Epub 2021 Feb 18.
Immunomodulation by mesenchymal stromal cells (MSCs) is a potentially important therapeutic modality. MSCs suppress peripheral blood mononuclear cell (PBMC) proliferation in vitro, suggesting a mechanism for suppressing inflammatory responses in vivo. This study details the interactions of PBMCs and MSCs.
Pooled human PBMCs and MSCs were co-cultured at different MSC:PBMC ratios and harvested from 0 to 120 h, with and without phytohaemagglutin A (PHA) stimulation. Proliferation of adherent MSCs and non-adherent PBMCs was assessed by quantitation of ATP levels. PBMC surface marker expression was analyzed by flow cytometry. Indoleamine 2,3-dioxygenase (IDO) activity was determined by kynurenine assay and IDO mRNA by RT-PCR. Cytokine release was measured by ELISA. Immunofluorescent microscopy detected MSC, PBMC, monocyte (CD14+) and apoptotic events.
PBMC proliferation in response to PHA gave a robust ATP signal by 72 h, which was suppressed by co-culture with densely plated MSCs. Very low level MSC seeding densities relative to PBMC number reproducibly stimulated PBMC proliferation. The CD4+/CD3+ population significantly decreased over time while the CD8+/CD3+ population significantly increased. No change in CD4+/CD8+ ratio is seen with high density MSC co-culture; very low density MSCs augment the changes seen in PHA stimulated PBMCs alone. IDO activity in MSCs co-cultured with PBMCs correlated with PBMC suppression. MSCs increased the secretion of IL-10 and IL-6 from stimulated co-cultures and decreased TNF-α secretion. In stimulated co-culture, low density MSCs decreased in number; fluorescence immunomicroscopy detected association of PBMC with MSC and phosphatidyl serine externalization in both cell populations.
A bidirectional interaction between MSCs and PBMCs occurs during co-culture. High numbers of MSCs inhibit PHA-stimulated PBMC proliferation and the PBMC response to stimulation; low numbers of MSCs augment these responses. Low density MSCs are susceptible to attrition, apparently by PBMC-induced apoptosis. These results may have direct application when considering therapeutic dosing of patients; low MSC doses may have unintended detrimental consequences.
间充质基质细胞（MSCs）的免疫调节作用是一种潜在的重要治疗方式。MSCs 在体外抑制外周血单个核细胞（PBMC）的增殖，这表明其在体内抑制炎症反应的机制。本研究详细描述了 PBMC 和 MSCs 之间的相互作用。
将混合的人 PBMC 和 MSCs 以不同的 MSC:PBMC 比例共培养，并在有或没有植物血球凝集素 A（PHA）刺激的情况下从 0 到 120 小时收获。通过定量测定 ATP 水平来评估贴壁 MSC 和非贴壁 PBMC 的增殖。通过流式细胞术分析 PBMC 表面标记物的表达。通过色氨酸酶测定法测定吲哚胺 2,3-双加氧酶（IDO）活性，并通过 RT-PCR 测定 IDO mRNA。通过 ELISA 测量细胞因子释放。免疫荧光显微镜检测 MSC、PBMC、单核细胞（CD14+）和凋亡事件。
PHA 刺激 PBMC 增殖在 72 小时内产生了强烈的 ATP 信号，而与密集接种的 MSC 共培养则抑制了该信号。相对于 PBMC 数量，极低密度的 MSC 播种密度可重复性地刺激 PBMC 增殖。随着时间的推移，CD4+/CD3+群体显著减少，而 CD8+/CD3+群体显著增加。高密度 MSC 共培养不会改变 CD4+/CD8+比值；极低密度 MSC 则增强了单独用 PHA 刺激 PBMC 时所观察到的变化。与 PBMC 共培养的 MSC 中的 IDO 活性与 PBMC 抑制相关。MSC 增加了刺激共培养物中 IL-10 和 IL-6 的分泌，并减少了 TNF-α的分泌。在刺激共培养物中，低密度 MSC 的数量减少；荧光免疫显微镜检测到 PBMC 与 MSC 的结合以及两种细胞群中磷脂酰丝氨酸的外化。
在共培养过程中，MSC 和 PBMC 之间发生了双向相互作用。大量 MSC 抑制 PHA 刺激的 PBMC 增殖和 PBMC 对刺激的反应；低数量的 MSC 增强了这些反应。低密度 MSC 易受损耗，显然是由 PBMC 诱导的凋亡引起的。这些结果在考虑患者的治疗剂量时可能具有直接的应用价值；低剂量的 MSC 可能会产生意想不到的有害后果。
