IRAK-4- and MyD88-dependent pathways are essential for the removal of developing autoreactive B cells in humans. Homo sapiens

Most autoreactive B cells are normally counterselected during early B cell development. To determine whether Toll-like receptors (TLRs) regulate the removal of autoreactive B lymphocytes, we tested the reactivity of recombinant antibodies from single B cells isolated from patients deficient for IL-1R-associated kinase (IRAK)-4, myeloid differentiation factor 88 (MyD88) and UNC-93B. Indeed, all TLRs except TLR3 require IRAK-4 and MyD88 to signal and UNC-93B-deficient cells are unresponsive to TLR3, TLR7, TLR8 and TLR9. All patients suffered from defective central and peripheral B cell tolerance checkpoints resulting in the accumulation of large numbers of autoreactive mature naïve B cells in their blood. Hence, TLR7, TLR8, and TLR9 may prevent the recruitment of developing autoreactive B cells in healthy donors. Paradoxically, IRAK-4-, MyD88- and UNC-93B-deficient patients did not display autoreactive antibodies in their serum nor developed autoimmune diseases, suggesting that IRAK-4, MyD88 and UNC-93B pathway blockade may thwart autoimmunity in humans. Overall design: RNA was extracted from 105-3.105 batch sorted new emigrant and mature naïve B cells isolated from donors using the Absolutely RNA microprep kit (Stratagene). 100-200 ng of RNA was obtained per sample, and the quality of the purified RNA was assessed by the Bioanalyzer from Agilent. Using the Ovation biotin system kit from Nugen, 30-50ng of RNA was amplified and labeled to produce cDNA. Labeled cDNA was hybridized on chips containing the whole human genome (Human Genome U133 2.0 from Affymetrix). Raw data from new emigrant (1 healthy donor) and mature naive (4 healthy donors) B cells were analyzed in order to determine the expression of some molecules involved in the TLR pathway in these B cell population in humans.

正常情况下，大多数自身反应性B细胞（autoreactive B cell）会在B细胞早期发育阶段被负向筛选清除。为探究Toll样受体（Toll-like receptors, TLRs）是否参与调控自身反应性B淋巴细胞的清除过程，我们对来自白细胞介素-1受体相关激酶（IL-1R-associated kinase, IRAK）-4、髓系分化因子88（myeloid differentiation factor 88, MyD88）及UNC-93B缺陷患者的单B细胞所产生的重组抗体反应性进行了检测。事实上，除TLR3外，所有TLR均依赖IRAK-4与MyD88进行信号转导；而UNC-93B缺陷细胞对TLR3、TLR7、TLR8及TLR9均无应答。上述患者均存在中枢与外周B细胞免疫检查点功能缺陷，导致其血液中大量蓄积自身反应性成熟初始B细胞（mature naïve B cell）。由此可见，TLR7、TLR8及TLR9可在健康供体内阻止发育中的自身反应性B细胞的募集。

矛盾的是，IRAK-4、MyD88及UNC-93B缺陷患者的血清中未检测到自身反应性抗体，也未发生自身免疫疾病，这提示阻断IRAK-4、MyD88及UNC-93B通路或可抑制人类自身免疫病的发生。

实验设计：我们使用Absolutely RNA微量制备试剂盒（Stratagene）从经批量分选的1×10^5~3×10^5个新近移民B细胞（new emigrant B cell）及成熟初始B细胞中提取总RNA，每份样本可获得100~200 ng纯化RNA，其质量通过安捷伦生物分析仪（Agilent Bioanalyzer）进行评估。采用Nugen公司的Ovation生物素标记系统试剂盒对30~50 ng RNA进行扩增与标记以制备cDNA。将标记后的cDNA与Affymetrix Human Genome U133 2.0全基因组芯片进行杂交。我们对来自1名健康供体的新近移民B细胞及4名健康供体的成熟初始B细胞的原始数据进行分析，以明确人类此类B细胞群体中参与TLR通路的部分分子的表达情况。