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).