Graduate Program in Immunology, Program in Biomedical Sciences and Division of Rheumatology, Department of Internal Medicine, University of Michigan Medical School, Ann Arbor, MI, USA.
Division of Rheumatology, Department of Internal Medicine, University of Michigan Medical School, Ann Arbor, MI, USA.
Methods Mol Biol. 2021;2270:149-178. doi: 10.1007/978-1-0716-1237-8_9.
B lymphocytes make several contributions to immune regulation including production of antibodies with regulatory properties, release of immune suppressive cytokines, and expression of death-inducing ligands. A role for Fas ligand (FasL)-expressing "killer" B cells in regulating T helper (T) cell survival and chronic inflammation has been demonstrated in animal models of schistosome worm and other infections, asthma, autoimmune arthritis, and type 1 diabetes. FasL B cells were also capable of inducing immune tolerance in a male-to-female transplantation model. Interestingly, populations of B cells found in the spleen and lungs of naïve mice constitutively expresses FasL and have potent killer function against T cells that is antigen-specific and FasL-dependent. Epstein-Barr virus-transformed human B cells constitutively express FasL and package it into exosomes that co-express MHC Class II molecules and have killer function against antigen-specific T cells. FasL exosomes with markers of B-cell lineage are abundant in the spleen of naïve mice. Killer B cells therefore represent a novel target for immune modulation in many disease settings. Our laboratory has published methods of characterizing FasL B cells and inducing their proliferation in vitro. This updated chapter will describe methods of identifying and expanding killer B cells from mice, detecting FasL expression in B cells, extracting FasL exosomes from spleen and culture supernatants, and performing functional killing assays against antigen-specific T cells.
B 淋巴细胞对免疫调节有多种贡献，包括产生具有调节特性的抗体、释放免疫抑制细胞因子以及表达诱导细胞死亡的配体。在血吸虫和其他感染、哮喘、自身免疫性关节炎和 1 型糖尿病的动物模型中，已经证明 Fas 配体（FasL）表达的“杀伤”B 细胞在调节辅助性 T（T）细胞存活和慢性炎症中发挥作用。FasL B 细胞还能够在雄性到雌性移植模型中诱导免疫耐受。有趣的是，在幼稚小鼠的脾脏和肺部中发现的 B 细胞群体持续表达 FasL，并具有针对 T 细胞的强大杀伤功能，这种杀伤功能是抗原特异性和 FasL 依赖性的。 Epstein-Barr 病毒转化的人 B 细胞持续表达 FasL，并将其包装成外泌体，这些外泌体共表达 MHC Ⅱ类分子，并具有针对抗原特异性 T 细胞的杀伤功能。幼稚小鼠脾脏中存在大量带有 B 细胞谱系标记的 FasL 外泌体。因此，杀伤 B 细胞代表了许多疾病情况下免疫调节的一个新靶点。我们的实验室已经发表了鉴定 FasL B 细胞并在体外诱导其增殖的方法。本章将更新描述从小鼠中鉴定和扩增杀伤 B 细胞、检测 B 细胞中 FasL 表达、从脾脏和培养上清液中提取 FasL 外泌体以及针对抗原特异性 T 细胞进行功能杀伤测定的方法。
