Park Gilseung, Foster Clay A, Malone-Perez Megan, Hasan Ameera, Macias Jose Juan, Frazer J Kimble
Department of Cell Biology, University of Oklahoma Health Sciences Center, Oklahoma City, OK.
Department of Pediatrics, Section of Pediatric Hematology-Oncology, University of Oklahoma Health Sciences Center, Oklahoma City, OK.
J Immunol. 2024 Dec 15;213(12):1902-1914. doi: 10.4049/jimmunol.2300818.
Zebrafish (Danio rerio) are a compelling model for studying lymphocytes because zebrafish and humans have similar adaptive immune systems, including their lymphocytes. Antibodies that recognize zebrafish proteins are sparse, so many investigators use transgenic, lymphocyte-specific fluorophore-labeled lines. Human and zebrafish lymphocyte types are conserved, but many aspects of zebrafish lymphocyte biology remain uninvestigated, including lymphocytes in peripheral tissues, like epidermis. This study is, to our knowledge, the first study to focus on zebrafish epidermal lymphocytes, using scales. Obtaining zebrafish blood via nonlethal methods is difficult; scales represent a source to longitudinally sample live fish. We developed a novel biopsy technique, collecting scales to analyze epithelial lymphocytes from several transgenic lines. We imaged scales via confocal microscopy and demonstrated multiple lymphocyte types in scales/epidermis, quantifying them flow cytometrically. We profiled gene expression of scale, thymic, and kidney-marrow (analogous to mammalian bone marrow) lymphocytes from the same animals, revealing B- and T-lineage signatures. Single-cell quantitative real-time PCR and RNA sequencing show not only canonical B and T cells but also novel lymphocyte populations not described previously. To validate longitudinal scale biopsies, we serially sampled scales from fish treated with dexamethasone, demonstrating epidermal lymphocyte responses. To analyze cells functionally, we employed a bead-ingestion assay, showing that thymic, marrow, and epidermal lymphocytes have phagocytic activity. In summary, we establish a novel, nonlethal technique to obtain zebrafish lymphocytes, providing the first quantification, expression profiling, and functional data from zebrafish epidermal lymphocytes.
斑马鱼（Danio rerio）是研究淋巴细胞的一种极具吸引力的模式生物，因为斑马鱼和人类具有相似的适应性免疫系统，包括淋巴细胞。识别斑马鱼蛋白质的抗体稀少，因此许多研究人员使用转基因的、淋巴细胞特异性荧光团标记的品系。人类和斑马鱼的淋巴细胞类型是保守的，但斑马鱼淋巴细胞生物学的许多方面仍未得到研究，包括外周组织（如表皮）中的淋巴细胞。据我们所知，本研究是第一项聚焦于斑马鱼表皮淋巴细胞的研究，使用鳞片进行研究。通过非致死性方法获取斑马鱼血液很困难；鳞片是纵向取样活鱼的一个来源。我们开发了一种新颖的活检技术，收集鳞片以分析来自多个转基因品系的上皮淋巴细胞。我们通过共聚焦显微镜对鳞片进行成像，并在鳞片/表皮中证实了多种淋巴细胞类型，通过流式细胞术对它们进行定量。我们分析了来自同一动物的鳞片、胸腺和肾髓质（类似于哺乳动物骨髓）淋巴细胞的基因表达，揭示了B细胞和T细胞谱系特征。单细胞定量实时PCR和RNA测序不仅显示了典型的B细胞和T细胞，还显示了以前未描述的新型淋巴细胞群体。为了验证纵向鳞片活检，我们对用 dexamethasone 处理的鱼的鳞片进行了连续取样，证实了表皮淋巴细胞的反应。为了从功能上分析细胞，我们采用了珠粒摄取试验，表明胸腺、骨髓和表皮淋巴细胞具有吞噬活性。总之，我们建立了一种新颖的、非致死性的技术来获取斑马鱼淋巴细胞，提供了来自斑马鱼表皮淋巴细胞的首次定量、表达谱分析和功能数据。
