Doctorate Program in Biological Sciences, Autonomous National University of Mexico, Mexico City, Mexico.
Immunodeficiency Laboratory, National Institute of Pediatrics, Mexico City, Mexico.
Front Immunol. 2024 Jul 15;15:1409434. doi: 10.3389/fimmu.2024.1409434. eCollection 2024.
Lipopolysaccharide-responsive and beige-like anchor (LRBA) is a scaffolding protein that interacts with proteins such as CTLA-4 and PKA, the importance of which has been determined in various cell types, including T regulatory cells, B cells, and renal cells. LRBA deficiency is associated with an inborn error in immunity characterized by immunodeficiency and autoimmunity. In addition to defects in T regulatory cells, patients with LRBA deficiency also exhibit B cell defects, such as reduced cell number, low memory B cells, hypogammaglobulinemia, impaired B cell proliferation, and increased autophagy. Although mice do not exhibit the immunodeficiency observed in humans, responses to B cell receptors (BCR) in B cells have not been explored. Therefore, a murine model is for elucidating the mechanism of Lrba mechanism in B cells.
To compare and evaluate spleen-derived B cell responses to BCR crosslinking in C57BL6 and mice.
Spleen-derived B cells were obtained from 8 to 12-week-old mice. Subpopulations were determined by immunostaining and flow cytometry. BCR crosslinking was assessed by the F(ab')2 anti-μ chain. Activation, proliferation and viability assays were performed using flow cytometry and protein phosphorylation was evaluated by immunoblotting. The nuclear localization of p65 was determined using confocal microscopy. Nur77 expression was evaluated by Western blot.
B cells showed an activated phenotype and a decreased proportion of transitional 1 B cells, and both proliferation and survival were affected after BCR crosslinking in the mice. The NF-κB pathway exhibited a basal activation status of several components, resulting in increased activation of p50, p65, and IκBα, basal p50 activation was reduced by the Plcγ2 inhibitor U73122. BCR crosslinking in B cells resulted in poor p50 phosphorylation and p65 nuclear localization. Increased levels of Nur77 were detected.
These results indicate the importance of Lrba in controlling NF-κB activation driven by BCR. Basal activation of NF-κB could impact cellular processes, such as, activation, differentiation, proliferation, and maintenance of B cells after antigen encounter.
脂多糖反应和米色样锚定（LRBA）是一种支架蛋白，可与 CTLA-4 和 PKA 等蛋白相互作用，其重要性已在包括 T 调节细胞、B 细胞和肾细胞在内的各种细胞类型中得到确定。LRBA 缺乏与一种以免疫缺陷和自身免疫为特征的先天性免疫错误有关。除了 T 调节细胞缺陷外，LRBA 缺乏症患者还表现出 B 细胞缺陷，例如细胞数量减少、记忆 B 细胞低、低丙种球蛋白血症、B 细胞增殖受损和自噬增加。虽然 小鼠没有表现出人类观察到的免疫缺陷，但尚未探索 B 细胞受体（BCR）在 B 细胞中的反应。因此，建立一种小鼠模型对于阐明 Lrba 在 B 细胞中的作用机制非常重要。
比较和评估 C57BL6 和 小鼠脾脏来源的 B 细胞对 BCR 交联的反应。
从 8 至 12 周龄的小鼠中获得脾脏来源的 B 细胞。通过免疫染色和流式细胞术确定亚群。通过 F(ab')2 抗 μ 链交联 BCR。通过流式细胞术和免疫印迹评估 BCR 交联后的激活、增殖和活力测定。通过共聚焦显微镜确定核内 p65 的定位。通过 Western blot 评估 Nur77 的表达。
BCR 交联后， 小鼠的 B 细胞表现出激活表型，过渡 1 B 细胞比例降低，增殖和存活均受到影响。NF-κB 途径的几个组成部分存在基础激活状态，导致 p50、p65 和 IκBα 的激活增加，Plcγ2 抑制剂 U73122 可降低基础 p50 激活。 小鼠的 BCR 交联导致 p50 磷酸化和 p65 核定位不良。检测到 Nur77 水平升高。
这些结果表明 Lrba 在控制 BCR 驱动的 NF-κB 激活中的重要性。NF-κB 的基础激活可能会影响细胞过程，例如抗原结合后 B 细胞的激活、分化、增殖和维持。
