Somatic deficiency of human CBL in leukocytes impairs B cell but not T cell development and function

The casitas B lineage lymphoma (CBL) proto-oncogene promotes positive selection and antigen responses in murine T lymphocytes by ubiquitinating ZAP70. However, CBL and CBL-B are redundant for ubiquitination of SYK and regulation of B cell receptor (BCR) signaling in murine B cells. We studied lymphocyte development, maturation and function in patients with somatic homozygosity for CBL loss-of-function variants in leukocytes. Surprisingly, human CBL is largely redundant for the development and function of human T cells. Conversely, CBL is critical for B cell development and function. Patients with somatic, hematopoietic CBL deficiency have a 10-fold increase in transitional B cells during childhood and are susceptible to bacterial infections. CBL deficiency impairs B cell maturation in a cell-intrinsic manner through reduced apoptosis and dysregulated BCR signaling, thereby reducing the proportions and numbers of memory B cells. Overall, our findings demonstrate that CBL deficiency has two major effects on human B cells. First, B-cell development in the bone marrow is altered at the immature stage, resulting in enhanced survival and differentiation of autoreactive B cell clones and impaired B cell tolerance, manifested as the production of autoantibodies. Second, the generation of antigen-specific B cells is impaired, thereby disrupting the establishment of adaptive immune memory. Thus, our study reveals the critical role of human CBL in B cells and its redundancy in T cells. Overall design: Bulk RNASeq on healthy donor and CBL LOH naïve B cells RNA was extracted with the RNeasy Plus Micro Kit (Qiagen) from sorted naïve B cells (2 × 104 cells) sorted from the PBMCs of CBL LOH patients, and age-matched healthy children. Full-length cDNA was generated from 1 ng total RNA with the SMART-Seq v4 Ultra Low Input RNA Kit (Clontech, 634888), and 1 ng cDNA was then used to prepare libraries with the Nextera XT DNA Library Preparation Kit (Illumina, FC-131-1024). Libraries with unique barcodes were pooled at equal molar ratios and sequenced on an Illumina NovaSeq 6000 sequencer with V1.5 reagents and NovaSeq Control Software V1.7.0 to generate 100 bp paired-end reads, according to the manufacturer's protocol. The FASTQ files generated were first inspected by fastqc to check sequencing quality. Sequences were aligned with the GENCODE reference genome (GRCh37.p13 and GRCm38.p6 for human and mouse, respectively) with STAR aligner v2.6. Gene-level features were quantified with featureCounts v1.6.0. Geneset enrichment analysis (GSEA) was conducted with the fgsea package by projecting genes ranked by their fold-changes in expression with effect-size shrinkage onto the Hallmark genesets retrieved from the MSigDB database (https://www.gsea-msigdb.org/gsea/msigdb/). Several of the healthy pediatric donor datasets are already published (68) and accessible on the NCBI Sequence Read Archive (accession no. PRJNA1141130).

卡斯汀B系淋巴瘤（casitas B lineage lymphoma, CBL）原癌基因可通过泛素化ζ链相关蛋白激酶70（ZAP70），促进小鼠T淋巴细胞的阳性选择与抗原应答。然而，在小鼠B细胞中，CBL与CBL-B在泛素化脾酪氨酸激酶（SYK）及调控B细胞受体（B cell receptor, BCR）信号通路方面功能冗余。
我们对白细胞中存在CBL功能丧失变异体的体细胞纯合性患者的淋巴细胞发育、成熟与功能进行了研究。令人意外的是，人类CBL在人T细胞的发育与功能中整体上呈现功能冗余性。与之相反，CBL对B细胞的发育与功能至关重要。
存在体细胞造血系统CBL缺陷的患者，儿童时期的过渡型B细胞数量会增加10倍，且易受细菌感染。CBL缺陷会通过减少细胞凋亡与扰乱BCR信号通路，以细胞自主性方式损害B细胞成熟，进而降低记忆B细胞的比例与数量。
综上，我们的研究结果表明，CBL缺陷对人B细胞存在两大主要影响。其一，骨髓中的B细胞发育在未成熟阶段发生异常，导致自身反应性B细胞克隆的存活与分化增强，同时B细胞耐受受损，表现为自身抗体的产生；其二，抗原特异性B细胞的生成受到损害，进而破坏适应性免疫记忆的建立。因此，本研究揭示了人类CBL在B细胞中的关键作用，以及其在T细胞中的功能冗余性。
整体实验设计：对健康供者与CBL杂合性缺失（loss of heterozygosity, LOH）患者的初始B细胞进行批量RNA测序（bulk RNASeq）。从CBL LOH患者与年龄匹配的健康儿童的外周血单个核细胞（peripheral blood mononuclear cell, PBMC）中分选得到初始B细胞（2×10⁴个细胞），使用RNeasy Plus Micro试剂盒（Qiagen）提取RNA。采用SMART-Seq v4超低起始量RNA试剂盒（Clontech, 634888），从1 ng总RNA中合成全长cDNA，随后取1 ng cDNA使用Nextera XT DNA文库制备试剂盒（Illumina, FC-131-1024）构建文库。将带有唯一条形码的文库按等摩尔浓度混合，按照厂商方案在Illumina NovaSeq 6000测序仪上使用V1.5试剂与NovaSeq控制软件V1.7.0进行测序，生成100 bp双端读长序列。
生成的FASTQ文件首先通过fastqc进行测序质量检测。使用STAR比对器v2.6，将序列比对至GENCODE参考基因组（人类为GRCh37.p13，小鼠为GRCm38.p6）。使用featureCounts v1.6.0对基因水平的特征进行定量。采用fgsea包进行基因集富集分析（gene set enrichment analysis, GSEA）：将经效应量收缩处理、按表达倍数变化排序的基因，投射至从MSigDB数据库（https://www.gsea-msigdb.org/gsea/msigdb/）获取的Hallmark基因集中。部分健康儿科供者的数据集已发表（文献68），可在NCBI序列读取档案库中获取，登录号为PRJNA1141130。