Somatic deficiency of human E3 ubiquitin ligase 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: Umbilical cord blood CD34+ hematopoietic stem and progenitor cells were edited at the AAVS1 safe harbor locus (n=2), the CBL locus (in frame exon 8 deletion; n =2) or base edited use ABEm8 at the PIK3CD locus (n=1) to generate the p.C416R gain-of-function variant. To initiate B cell differentiation, the edited CD34+ cells were co-cultured with MS-5 stroma cells in the presence of IL-7. At day 21 of co-culture (day 24 post editing), the cells were collected from the MS5, sorted for CD19+CD10+CD20dim and subsequently rested for 12 hours in RPMI+10%FBS. RNA was extracted from each sample in duplicates using the Total RNA Purification Micro Kit (Norgen, #35300) according to the manufacturer's instructions. All samples were treated with DNase I to remove residual genomic DNA.