Data_Sheet_1_Characterization of Phenotypes and Functional Activities of Leukocytes From Rheumatoid Arthritis Patients by Mass Cytometry.PDF

Background: Rheumatoid arthritis (RA) is the most common autoimmune rheumatic disease and leads to persistent chronic inflammation. The pathophysiology of the disease is complex, involving both adaptive and innate immunity. Among innate immune cells, neutrophils have been rarely studied due to their sensitivity to freezing and they are not being collected after Ficoll purification.
Methods: We used mass cytometry to perform a multidimensional phenotypic characterization of immune cells from RA-treated patients, which included the simultaneous study of 33 intra- or extra-cellular markers expressed by leukocytes. We were able to focus our study on innate immune cells, especially neutrophils, due to a specific fixation method before freezing. In addition, blood samples were stimulated or not with various TLR agonists to evaluate whether RA-dependent chronic inflammation can lead to immune-cell exhaustion.
Results: We show that RA induces the presence of CD11blow neutrophils (33.7 and 9.2% of neutrophils in RA and controls, respectively) associated with the duration of disease. This subpopulation additionally exhibited heterogeneous expression of CD16. We also characterized a CD11ahigh Granzyme Bhigh T-cell subpopulation possibly associated with disease activity. There was no difference in cytokine expression after the stimulation of immune cells by TLR agonists between RA and controls.
Conclusion: Mass cytometry and our fixation method allowed us to identify two potential new blood subpopulations of neutrophils and T-cells, which could be involved in RA pathology. The phenotypes of these two potential new subpopulations need to be confirmed using other experimental approaches, and the exact role of these subpopulations is yet to be studied.

背景：类风湿关节炎（Rheumatoid arthritis, RA）是最常见的自身免疫性风湿性疾病，可引发持续性慢性炎症。该疾病的病理生理学机制复杂，涉及适应性免疫与固有免疫两大分支。在固有免疫细胞中，中性粒细胞因对冻存极为敏感，且无法通过菲科（Ficoll）纯化法获取，相关研究长期以来较为匮乏。
方法：本研究采用质谱流式细胞术（mass cytometry），对接受RA治疗的患者体内免疫细胞进行多维度表型表征，同步检测白细胞表达的33种细胞内及细胞外标志物。得益于冻存前采用的特异性固定方法，本研究得以聚焦固有免疫细胞，尤其是中性粒细胞。此外，我们将部分血液样本予以多种Toll样受体激动剂（TLR agonists）刺激，其余样本不予处理，以此探究RA相关的慢性炎症是否会引发免疫细胞耗竭。
结果：本研究发现，RA可诱导CD11b低表达（CD11blow）中性粒细胞的产生，在RA患者组与对照组的中性粒细胞中占比分别为33.7%与9.2%，且该细胞亚群的占比与疾病病程呈相关性。此亚群同时表现出CD16表达的异质性。此外，我们还鉴定出一类CD11a高表达、颗粒酶B高表达（CD11ahigh Granzyme Bhigh）的T细胞亚群，其可能与疾病活动度相关。在使用Toll样受体激动剂刺激免疫细胞后，RA患者组与对照组的细胞因子表达水平无显著差异。
结论：质谱流式细胞术结合本研究采用的特异性固定方法，使我们得以鉴定出两类潜在的新型血液免疫细胞亚群——中性粒细胞亚群与T细胞亚群，它们可能参与RA的病理进程。这两类新型亚群的表型需通过其他实验手段进一步验证，其确切的病理生理学角色仍有待深入研究。