Research in chickens has been fundamental for the discovery of basic aspects of the immune system and has led to an interest in the in-depth characterization of avian immune cell types including dendritic cells (DCs). The in vitro generation and expansion of chicken bone marrow-derived DCs (chBMDCs) in the presence of granulocyte-macrophage colony-stimulating factor (GM-CSF) has provided a way to study chicken DCs, which are only present at limited cell numbers in vivo. This method has been employed to study the interactions between chicken DCs and pathogens or vaccines. However, a detailed characterization of the chBMDC culture is still lacking. In the present study, we performed an elaborate phenotypical and functional analysis of the chBMDC culture and addressed its heterogeneity. After 8 days of culture, chBMDCs comprised major histocompatibility complex class II (MHC-II)^low and MHC-II^high subsets with different morphologies. Compared with MHC-II^low chBMDCs, the MHC-II^high subset showed a more mature phenotype, with higher expressions of CD1.1, CD40, CD80, CCR7, and CD83, and a relatively low opsonophagocytic capacity. Nevertheless, MHC-II^high chBMDCs did not show an increased capacity to induce T-cell proliferation. Therefore, MHC-II^high chBMDCs were found to be semi-mature. Interestingly, the presence of the semi-mature MHC-II^high chBMDC subset reduced when cells were cultured in the presence of IL-4. Finally, prolonged cell culture after fluorescence-activated cell sorting (FACS) converted the semi-mature MHC-II^high subset back into the immature phenotype of the MHC-II^low subset, demonstrating plasticity of their maturation state. This detailed characterization explained the heterogeneity of the chBMDC culture by the simultaneous presence of immature and semi-mature chBMDC subsets, in addition to cells without features of antigen-presenting cells. Our findings are instrumental for the interpretation of experiments using the chBMDC culture in past and future research by providing insights into its phenotypically and functionally distinct cell types.

鸡的研究一直是发现免疫系统基本方面的基础，并引起了对包括树突状细胞（DC）在内的禽类免疫细胞类型的深入表征的兴趣。的体外 粒细胞-巨噬细胞集落刺激因子（GM-CSF）存在下鸡骨髓来源DC（chBMDC）的产生和扩增提供了研究仅存在于有限细胞数量的鸡DC的方法 体内。该方法已用于研究鸡DC与病原体或疫苗之间的相互作用。但是，仍然缺乏chBMDC文化的详细特征。在本研究中，我们对chBMDC文化进行了详尽的表型和功能分析，并探讨了其异质性。培养8天后，chBMDCs包含主要的组织相容性复合物II类（MHC-II）^低和MHC-II^高子集，具有不同的形态。与MHC-II相比^低chBMDCs时，MHC-II^的高子组显示出更成熟的表型，与CD1.1，CD40，CD80，CCR7，以及CD83，和相对低的调理吞噬能力的更高表达。尽管如此，MHC-II^高chBMDC没有显示出增强的诱导T细胞增殖的能力。因此，发现MHC-II^高chBMDC是半成熟的。有趣的是，当在IL-4存在下培养细胞时，半成熟MHC-II^高chBMDC亚群的存在减少了。最后，经过荧光激活细胞分选（FACS）后的长时间细胞培养将半成熟的MHC-II^高子集转化回MHC-II^低的未成熟表型子集，说明其成熟状态的可塑性。除了没有抗原呈递细胞特征的细胞外，这种详细的特征还通过同时存在未成熟和半成熟的chBMDC亚群来解释chBMDC培养的异质性。我们的发现有助于通过对chBMDC文化在表型和功能上不同的细胞类型的深入了解来解释在过去和将来的研究中使用chBMDC文化进行的实验。